Therapeutic vitamin D conjugates

ABSTRACT

The invention provides non-hormonal vitamin D conjugated to apelin proteins that result in increased absorption, bioavailability or circulating half-life when compared to non-conjugated forms. In some embodiments, the vitamin D targeting groups are coupled to the apelin proteins via the third carbon on the vitamin D backbone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/244,181 filed Oct. 20, 2015 and U.S. Provisional Application No.62/067,388 filed Oct. 22, 2014, the contents of which are incorporatedby reference herein in their entirety.

This invention was made with Government support under Grant No.IIP-1430894 awarded by the National Science Foundation, and Grant Nos.1-R43-CA174094-01A1 and 1-R43-DK107231-01A1 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 5, 2015, isnamed XTND005US1_SL.txt and is 17,869 bytes in size.

FIELD OF THE INVENTION

The invention provides non-hormonal vitamin D conjugated to therapeuticcompounds that result in the compounds having increased absorption,bioavailability or circulating half-life when compared to non-conjugatedforms. The vitamin D targeting groups are coupled to the therapeuticcompounds via the third carbon on the vitamin D backbone.

BACKGROUND OF THE INVENTION

The invention relates to improving the potency, absorption orpharmacokinetic properties of therapeutic compounds to certain vitamin Dforms. Vitamin D plays a role in calcium, phosphate, and bonehomeostasis. The hormonal activity of vitamin D is mediated throughbinding to the vitamin D receptor (VDR). It enters the nucleus where itbinds to the vitamin D receptor element (VDRE) present in the promotersof a subset of genes that are thus responsive to hormonal Vitamin D.

Vitamin D is a group of fat-soluble secosteroids. Several forms(vitamers) of vitamin D exist. The two major forms are vitamin D2 orergocalciferol, and vitamin D3 or cholecalciferol. Vitamin D without asubscript refers to vitamin D2, D3 or other forms known in the art. Inhumans, vitamin D can be ingested as cholecalciferol (vitamin D3) orergocalciferol (vitamin D2). The major source of vitamin D for mosthumans is sunlight. Once vitamin D is made in the skin or ingested, itneeds to be activated by a series of hydroxylation steps, first to25-hydroxyvitamin D (25(OH)D3) in the liver and then to1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the kidney. 1α,25(OH)2D3 isthe active “hormonal” form of vitamin D because it binds to VDR.25(OH)D3 is the “non-hormonal” form of vitamin D and is the majorcirculating form in the human body. It binds the vitamin D BindingProtein (DBP). It is only converted to the hormonal form as needed. Anexample of a non-hormonal vitamin D form is one that lacks a la-hydroxylgroup. Non-hormonal vitamin D forms have a greatly reduced affinity forVDR and a greatly increased affinity for DBP.

DBP is the principal transporter of vitamin D metabolites. Itsconcentration in the plasma is 6-7 μM and has been detected in all fluidcompartments. DBP concentrations exceed the physiological vitamin Dmetabolite concentrations. DBP is important for the translocation ofvitamin D from the skin into circulation, and across cell membranes intothe cytoplasm where vitamin D is activated into the hormonal form. Theaffinity of non-hormonal Vitamin D for DBP is significantly higher thanthe affinity of the hormonal form. In contrast, the affinity of thehormonal form to VDR is significantly than the non-hormonal form.

Vitamin D and vitamin D analogs have been approved for the treatment ofosteoporosis and secondary hyperparathyroidism. Vitamin D has also beenshown to inhibit proliferation and induce differentiation in normal aswell as cancer cells. The level of vitamin D required for this activitycauses severe toxicity in the form of hypercalcemia. Analogs of vitaminD have been approved for the treatment of psoriasis and others arecurrently being tested for cancer treatment. Many of the analogsdiscovered to have a reduced calcemic effect contain side-chainmodifications. These modifications do not greatly affect VDR binding,and thus, in cell-based proliferation assays, show equal or evenincreased efficacy. It was shown, however, that many of thesemodifications reduce binding to DBP and thereby reduce the half-life inthe bloodstream.

The addition of poly(ethylene glycol) or (PEG) is a known method ofincreasing the half-life of some compounds by reducing kidney clearance,reducing aggregation, and diminishing potentially unwanted immunerecognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403-447(2008)). The PEG is typically used at a considerably large size (20-40kDa) to maximize the half-life in circulation. This can be accomplishedby using either a single large PEG or multiple smaller PEGs attached tothe compound. (Clark et al. J. Biol. Chem. 271:21969-21977 (1996);Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)).

Absorption is a primary focus in drug development and medicinalchemistry because a drug must be absorbed before any medicinal effectscan take place. A drug's absorption profile can be affected by manyfactors. Additionally, the absorption properties of therapeuticcompounds vary significantly from compound to compound. Some therapeuticcompounds are poorly absorbed following oral or dermal administration.Other therapeutic compounds, such as most peptide- and protein-basedtherapeutics, cannot be administered orally. Alternate routes ofadministration such as intravenous, subcutaneous, or intramuscularinjections are routinely used for some of these compounds; however,these routes often result in slow absorption and exposure of thetherapeutic compounds to enzymes that can degrade them, thus requiringmuch higher doses to achieve efficacy.

A number of peptides have been identified as therapeutically promising.The chemical and biological properties of peptides and proteins makethem attractive candidates for use as therapeutic compounds. Peptidesand proteins are naturally-occurring molecules made up of amino acidsand are involved in numerous physiological processes. Peptides andproteins display a high degree of selectivity and potency, and may notsuffer from potential adverse drug-drug interactions or other negativeside effects. Thus peptides and proteins hold great promise as a highlydiverse, highly potent, and highly selective class of therapeuticcompounds with low toxicity. Peptides and proteins, however, may haveshort in vivo half-lives. For such peptides, this may be a few minutes.This may render them generally impractical, in their native form (alsoreferred to as “wild”, “wild type” or “wt” herein), for therapeuticadministration. Additionally, peptides may have a short duration ofaction or poor bioavailability.

Apelin peptide (SEQ ID NO:1) is encoded by the APLN gene. The apelingene encodes a pre-proprotein of 77 amino acids with a signal peptide inthe N-terminal region. After translocation into the endoplasmicreticulum and cleavage of the signal peptide, the proprotein of 55 aminoacids may generate several active fragments: a 36 amino acid peptidecorresponding to the sequence 42-77 (apelin 36), a 17 amino acid peptidecorresponding to the sequence 61-77 (apelin 17) and a 13 amino acidpeptide corresponding to the sequence 65-77 (apelin 13). This latterfragment may also undergo a pyroglutamylation at its N-terminalglutamine residue.

Apelin is the endogenous ligand for the G-protein-coupled APJ receptorthat is expressed at the surface of some cell types. It is widelyexpressed in various organs such as the heart, lung, kidney, liver,adipose tissue, gastrointestinal tract, brain, adrenal glands,endothelium, and human plasma.

The apelin receptor participates in the control of blood pressure andthe formation of new blood vessels (angiogenesis). Apelin causeshypotension from the activation of its receptors on the surface ofendothelial cells. This induces the release of NO, a potent vasodilator,which induces relaxation of the smooth muscle cells of artery wall. Theangiogenic activity results from Apelin promoting the proliferation andmigration of endothelial cells and the formation of new blood vessels.Other effects of apelin include regulation of fluid homeostasis,hypothalamic regulation of food and water intake, pituitary hormonerelease, and down-regulation of the antidiuretic hormone vasopressin inthe brain. Additionally, apelin is secreted in the gastrointestinaltract and in the pancreas.

Apelin regulates cardiovascular and fluid homeostasis, food intake, cellproliferation, and angiogenesis. Apelin is also considered to be anadipokine that is linked to metabolic disorders such as obesity and type2 diabetes. Apelin therapies may thus be a beneficial treatment forthese conditions. See, e.g., Castan-Laurell, et al., Endocrine 40(1):1-9(2011). Indeed, Apelin inhibits insulin secretion induced by glucose.Likewise, insulin stimulates apelin, revealing a feedback loop forinsulin production. The in vivo half-life of apelin, however, is 20minutes or less. See, e.g., Bertrand et al. Front Physiol. 6:115 (2015).

Ghrelin is a mammalian peptide (SEQ ID NO:2) that is naturally secretedfrom the stomach into circulation to stimulate appetite and release ofgrowth hormone (GH). Ghrelin stimulates the release of growth hormonefrom the pituitary gland through the cellular receptor GHS-R and playsan important role in energy homeostasis. In addition, ghrelin actsdirectly on the central nervous system to decrease sympathetic nerveactivity. GHS-Rs are concentrated in the hypothalamus-pituitary unit.GHS-R is distributed in peripheral tissues, including the heart, lung,liver, kidney, pancreas, stomach, small and large intestines, adipose,and immune cells.

Ghrelin has been used therapeutically to increase weight and lean bodymass in patients suffering from cachexia or involuntary weight lossresulting from chronic diseases such as cancer (Hiura et. al., Cancer,118:4785-94 (2012)). Ghrelin, however, has a naturally short half-lifeof 11 minutes in humans (Akamizu et al., Eur J Endocrinol 150:447-55(2004)) and thus must be dosed often to see therapeutic effects.

Parathyroid hormone (PTH), parathormone or parathyrin, is secreted bythe chief cells of the parathyroid glands. It is a polypeptidecontaining 84 amino acids (SEQ ID NO:10). It acts to increase theconcentration of calcium (Ca²⁺) in the blood (in contrast to calcitoninwhich decreases calcium concentration). PTH activates the parathyroidhormone 1 receptor (bone and kidney) and the parathyroid hormone 2receptor (central nervous system, pancreas, testis, and placenta). PTH,however, has a very short half-life of approximately 4 minutes.

Hypoparathyroidism is a low level of PTH in the blood that is mostcommonly due to damage to or removal of parathyroid glands duringthyroid surgery, immune system-related damage, inheritance, or otherrare causes. It can lead to low levels of calcium in the blood, oftencausing cramping and twitching of muscles or tetany (involuntary musclecontraction), and several other symptoms. Calcium replacement or vitaminD can ameliorate the symptoms but can increase the risk of kidney stonesand chronic kidney disease. See, e.g. Winer K K, et. al. J. Clin.Endocrinol. Metab. 97(2): 391-399 (2012).

Insulin is a peptide hormone produced by beta cells in the pancreas thatregulates the metabolism of carbohydrates and fats (SEQ ID NO:11 and12). The human insulin protein is composed of 51 amino acids, and has amolecular weight of 5808 Daltons. It is a dimer of an A-chain and aB-chain that are linked by disulfide bonds. It promotes the absorptionof glucose from the blood to skeletal muscles and fat tissue and causesfat to be stored rather than used for energy.

Under normal physiological conditions, insulin is produced at a constantproportion to remove excess glucose from the blood. When control ofinsulin levels fails, however, diabetes mellitus can result. Thus,diabetic patients often receive injected insulin. Patients with type 1diabetes depend on external insulin for their survival because thehormone is no longer sufficiently produced internally. Insulin is mostcommonly injected subcutaneously. Patients with type 2 diabetes areoften insulin resistant and may suffer from an “apparent” insulindeficiency.

Fibroblast Growth Factor 21 (SEQ ID:2) is a protein that circulates inserum. Encoded by the FGF21 gene, it is a member of a family of atypicalfibroblast growth factors (FGFs) that includes FGF19 and FGF23. It lacksthe conventional FGF heparin-binding domain. FGF family members possessbroad mitogenic and cell survival activities and are involved in avariety of biological processes including embryonic development, cellgrowth, morphogenesis, tissue repair, tumor growth and invasion. FGF21is specifically induced by HMGCS2 activity. FGF21 stimulates glucoseuptake in adipocytes but not in other cell types. This effect isadditive to the activity of insulin.

FGF21 prefers binding to the FGFR1c/b-Klotho receptor complex over thosecontaining other FGFR isotypes (Kliewer and Mangelsdorf, Am. J. Clin.Nutr. 91:254S-257S (2010)). Administration of FGF21 to diabetic animalsreduces circulating glucose levels while excess FGF21 does not inducehypoglycemia as seen with administration of excess insulin(Kharitonenkov and Shanafelt, Curr. Opin. Investig. Drugs 10:359-364(2009)). Therefore, FGF21 is a promising therapeutic protein for thetreatment of diabetes. FGF21 in its natural state, however, has anextremely short half-life in serum (about 1.1 hours) making it aclinically impractical treatment (see, e.g. WO03/011213; Kharitonenkovet al., J. Clin. Invest. 115:1627-1635 (2005)). Additionally, FGF21exhibits poor bioavailability when injected subcutaneously (Xu J et al.,2009. Am J Physiol. Endocrinol. Metab. 297: E1105-E1114).

Infliximab (Remicade®, Janssen Biotech Inc., U.S. Pat. No. 5,919,452 andUS 2002/0141996, incorporated herein by reference in their entirety) isa monoclonal antibody that binds tumor necrosis factor alpha (TNF-α, SEQID NO:13) that is used to treat autoimmune diseases. Infliximab wasapproved by the U.S. Food and Drug Administration (FDA) for thetreatment of psoriasis, Crohn's disease, ankylosing spondylitis,psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis. TNF-αis a chemical messenger (cytokine) and a key part of the autoimmunereaction. Infliximab is administered intravenously by a healthcareprofessional and is not approved for subcutaneous dosing.

RNA interference (RNAi) is a process where RNA molecules inhibit geneexpression often by causing specific mRNA molecules to degrade. Twotypes of RNA molecules—microRNA (miRNA) and small interfering RNA(siRNA)—are central to RNA interference. They bind to the target mRNAmolecules and either increase or decrease their activity. RNAi helpscells defend against parasitic nucleic acids such as those from virusesand transposons. RNAi also influences development.

Initial medical applications for RNAi involve genetic diseases such asmacular degeneration and Huntington's disease. Additional applicationsmay include certain cancers, respiratory syncytial virus, herpes simplexvirus type 2, HIV, hepatitis A and B, influenza, and measles.

It remains difficult to deliver RNAi to target tissues, and inparticular, tissues deep within the body. siRNA molecules have a shortin vivo half-life due to endogenous nucleases. Also, targeting specifictissues is challenging. One approach has been high dosage levels ofsiRNA to ensure the tissues have been reached. With these approaches,however, hepatotoxicity was reported.

Therapeutic oligonucleotides, while promising, suffer from a shortplasma half-life as well as from problems with delivery and cellularuptake. Conjugation of oligonucleotides to small molecules has beenproposed to overcome these problems but have not yet been successful.

SUMMARY OF THE INVENTION

The invention provides carrier-drug conjugates comprising a targetinggroup that is non-hormonal vitamin D, an analog, or metabolite thereoflinked at the carbon 3 position to a therapeutic compound. In someembodiments, the non-hormonal vitamin D molecules are not hydroxylatedat the carbon 1 position. The carriers enhance the absorption,stability, half-life, duration of effect, potency, or bioavailability ofthe therapeutic compounds. Optionally, the carriers further comprisescaffolding moieties that are non-releasable such as PEG and othersdescribed in this disclosure.

Thus, the invention provides a carrier-drug conjugate comprising atargeting group that is a non-hormonal vitamin D, analog, or metabolitethereof conjugated to a therapeutic compound at the carbon 3 position ofsaid non-hormonal vitamin D targeting group. In some embodiments, thenon-hormonal vitamin D is not hydroxylated at the carbon 1 position. Inpreferred embodiments, the targeting group is conjugated to thetherapeutic compound via a scaffold that is between about 100 and200,000 Da and is selected from the group consisting of poly(ethyleneglycol), polylysine, polyethyleneimine, poly(propyleneglycol), apeptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, anucleic acid, a glycan, a modifying group that contains a reactivelinker, a water-soluble polymer, a small carbon chain linker, and anadditional therapeutic compound.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising a carrier-drug conjugate comprising a targetinggroup that is a non-hormonal vitamin D, analog, or metabolite thereofconjugated to a therapeutic compound at the carbon 3 position of thenon-hormonal vitamin D targeting group via a scaffold. In a preferredembodiment, the carrier increases the absorption, bioavailability, orhalf-life of said therapeutic compound in circulation. In anotherpreferred embodiment, the non-hormonal vitamin D is not hydroxylated atthe carbon 1 position. In another preferred embodiment of thepharmaceutical composition, the scaffold is selected from the groupconsisting of poly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic compound.

In another preferred embodiment, the therapeutic compound is selectedfrom the group consisting of small molecules, chemical entities, nucleicacids, nucleic acid derivatives, peptides, peptide derivatives,naturally-occurring proteins, non-naturally-occurring proteins,peptide-nucleic acids (PNA), stapled peptides, morpholinos,phosphorodiamidate morpholinos, oligonucleotides, antisense drugs,RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones,cytokines, interferons, growth factors, blood coagulation factors,antibodies, antibody fragments, antibody derivatives, toxin-conjugatedantibodies, antibody-drug conjugates, metabolic effectors, analgesics,antipyretics, anti-inflammatory agents, antibiotics, anti-microbialagents, anti-viral agents, anti-fungal drugs, musculoskeletal drugs,cardiovascular drugs, renal drugs, pulmonary drugs, digestive diseasedrugs, hematologic drugs, urologic drugs, metabolism drugs, hepaticdrugs, neurological drugs, anti-diabetes drugs, anti-cancer drugs, drugsfor treating stomach conditions, drugs for treating colon conditions,drugs for treating skin conditions, and drugs for treating lymphaticconditions.

In a more preferred embodiment, the therapeutic compound is a proteinhaving apelin activity comprising an amino acid sequence with at least a90% sequence identity to SEQ ID NO:1 or 16. In another more preferredembodiment, the targeting group is vitamin D that is not hydroxylated atthe carbon 1 position. In another more preferred embodiment, thescaffold is poly(ethylene glycol).

The invention provides that the therapeutic compound may be a proteinhaving ghrelin activity comprising an amino acid sequence with at leasta 90% sequence identity to a protein selected from the group consistingof SEQ ID NO:2, 3, 4, and 5. In a more preferred embodiment, thetargeting group is vitamin D that is not hydroxylated at the carbon 1position. In other more preferred embodiments, the therapeutic compoundis a protein comprising the amino acid sequence of SEQ ID NO:2, 3, 4, or5. In another more preferred embodiment, the scaffold is poly(ethyleneglycol).

The invention provides a pharmaceutical composition comprising a proteinhaving PTH activity that has an amino acid sequence with at least a 90%sequence identity to SEQ ID NO:10 or 17. In a preferred embodiment, thetargeting group is vitamin D that is not hydroxylated at the carbon 1position. In another preferred embodiment, the scaffold is poly(ethyleneglycol).

The invention provides a pharmaceutical composition comprising a proteinhaving insulin activity and comprising a peptide having amino acid ansequences with at least a 90% sequence identity to SEQ ID NO:11 or 12.In a preferred embodiment, the targeting group is vitamin D that is nothydroxylated at the carbon 1 position. In another preferred embodiment,the scaffold is poly(ethylene glycol).

In one embodiment of the invention, the therapeutic compound is anantibody. In a preferred embodiment, the antibody binds with highaffinity to a protein having at least a 90% sequence identity to SEQ IDNO:13. In another preferred embodiment, the targeting group is vitamin Dthat is not hydroxylated at the carbon 1 position. In another preferredembodiment, the scaffold is poly(ethylene glycol).

The invention contemplates that the therapeutic compound is an RNAmolecule. In a preferred embodiment, the targeting group is vitamin Dthat is not hydroxylated at the carbon 1 position. In another preferredembodiment, the scaffold is poly(ethylene glycol).

The invention provides a method of treating a patient in need of atherapeutic compound, comprising administering an effective amount ofthe pharmaceutical compositions described herein. In some embodiments,the therapeutic compound is selected from the group consisting of smallmolecules, chemical entities, nucleic acids, nucleic acid derivatives,peptides, peptide derivatives, naturally-occurring proteins,non-naturally-occurring proteins, peptide-nucleic acids (PNA), stapledpeptides, morpholinos, oligonucleotides, morpholinos, antisense drugs,RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones,cytokines, interferons, growth factors, blood coagulation factors,antibodies, antibody fragments, antibody derivatives, toxin-conjugatedantibodies, antibody-drug conjugates, metabolic effectors, analgesics,antipyretics, anti-inflammatory agents, antibiotics, anti-microbialagents, anti-viral agents, anti-fungal drugs, musculoskeletal drugs,cardiovascular drugs, renal drugs, pulmonary drugs, digestive diseasedrugs, hematologic drugs, urologic drugs, metabolism drugs, hepaticdrugs, neurological drugs, anti-diabetes drugs, anti-cancer drugs, drugsfor treating stomach conditions, drugs for treating colon conditions,drugs for treating skin conditions, and drugs for treating lymphaticconditions.

In one embodiment of the method, therapeutic compound is a proteinhaving apelin activity comprising an amino acid sequence with at least a90% sequence identity to SEQ ID NO:1 or 15. In a preferred embodiment,the targeting group is vitamin D that is not hydroxylated at the carbon1 position. In another preferred embodiment, the scaffold ispoly(ethylene glycol).

In another embodiment of the method, the therapeutic compound is aprotein having ghrelin activity comprising an amino acid sequence withat least a 90% sequence identity to a protein selected from the groupconsisting of SEQ ID NO:2, 3, 4, and 5. In a preferred embodiment, thetargeting group is vitamin D that is not hydroxylated at the carbon 1position. In other preferred embodiments, the therapeutic compound is aprotein comprising the amino acid sequence of SEQ ID NO:2, 3, 4, or 5.In another preferred embodiment, the scaffold is poly(ethylene glycol).

In another embodiment of the method, the therapeutic compound is aprotein having PTH activity comprising an amino acid sequence with atleast a 90% sequence identity to SEQ ID NO:10 or 16. In a preferredembodiment, the targeting group is vitamin D that is not hydroxylated atthe carbon 1 position. In another preferred embodiment, the scaffold ispoly(ethylene glycol).

In another embodiment of the method, the therapeutic compound is aprotein having insulin activity comprising an amino acid sequence withat least a 90% sequence identity to SEQ ID NO:11 or at least a 90%sequence identity to SEQ ID NO:12. In a preferred embodiment, thetargeting group is vitamin D that is not hydroxylated at the carbon 1position. In another preferred embodiment, the scaffold is poly(ethyleneglycol).

In another embodiment of the method, the therapeutic compound is anantibody. In a preferred embodiment, the antibody binds with highaffinity to a protein having at least a 90% sequence identity to SEQ IDNO:13. In another preferred embodiment, the targeting group is vitamin Dthat is not hydroxylated at the carbon 1 position. In another preferredembodiment, the scaffold is poly(ethylene glycol).

In another embodiment of the method, the therapeutic compound is an RNAmolecule. In a preferred embodiment, the targeting group is vitamin Dthat is not hydroxylated at the carbon 1 position. In another preferredembodiment, the scaffold is poly(ethylene glycol).

The methods of the invention provide that the pharmaceuticalcompositions are delivered to patients by a transdermal, oral,parenteral, subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intrasynovial, intrasternal, intrathecal, intralesional,intracranial injection, infusion, inhalation, ocular, topical, rectal,nasal, buccal, sublingual, vaginal, or implanted reservoir mode.

The invention provides pharmaceutical compositions for the manufactureof a medicament for the treatment of a patient in need of saidmedicament.

The invention provides a method of manufacturing the pharmaceuticalcomposition disclosed herein, comprising conjugating the targeting groupand the therapeutic compound, wherein the conjugating step utilizes acoupling group. In preferred embodiments, the coupling group is selectedfrom the group consisting of an amine-reactive group, a thiol-reactivegroup, a maleimide group, a thiol group, an aldehyde group, anNETS-ester group, a haloacetyl group, an iodoacetyl group, a bromoacetylgroups, a SMCC group, a sulfo SMCC group, a carbodiimide group,bifunctional cross-linkers, NHS-maleimido, and combinations thereof.Thus, the invention provides pharmaceutical compositions resulting fromthe methods, wherein the composition comprises a carrier-drug compoundcontaining a linkage selected from the group consisting of a thiollinkage, an amide linkage, an oxime linkage, a hydrazone linkage, and athiazolidinone linkage. In another embodiment, the conjugating step isaccomplished by cycloaddition reactions.

The invention provides a pharmaceutical carrier comprising a formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:B is a targeting group that is a non-hormonal vitamin D, analog, ormetabolite thereof conjugated at the carbon 3 position to L¹;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, polylactic acid, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic moiety;C is an amine-reactive group, a thiol-reactive group, a maleimide group,a thiol group, a disulfide group, an aldehyde group, an NETS-estergroup, a 4-nitrophenyl ester, an acylimidazole, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido or combinations thereof;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

The invention provides a pharmaceutical carrier comprising formula V:

The invention provides a pharmaceutical carrier comprising formula VI:

The invention provides a pharmaceutical carrier comprising formula VII:

The invention provides a pharmaceutical composition, comprising atherapeutic compound, a stably attached scaffold, a targeting group thatis a non-hormonal vitamin D, analog, or metabolite thereof conjugated atthe carbon 3 position, wherein after administration to a first testsubject, the therapeutic compound has a half life measured by ELISAanalysis of blood samples taken at a plurality of time points that isgreater than a half life of the therapeutic compound administered to asecond test subject without the stably attached scaffold moiety andtargeting group as measured by ELISA analysis of blood samples taken atthe plurality of time points. In a preferred embodiment, theadministration to the first and second subjects is accomplished bysubcutaneous injection. In another preferred embodiment, the therapeuticcompound stably attached to the scaffold and targeting group retainssubstantially the same activity as the therapeutic compound not stablyattached to the scaffold and targeting group as measured by a functionalassay.

In another preferred embodiment of the pharmaceutical composition, ascaffold mass range is selected from the group consisting of 100 Da. to20,000 Da., 200 Da. to 15,000 Da., 300 Da. to 10,000 Da., 400 Da. to9,000 Da., 500 Da. to 5,000 Da., 600 Da. to 2,000 Da., 1000 Da. to200,000 Da., 20,000 Da. to 200,000 Da., 100,000 to 200,000 Da., 5000 Da.to 100,000 Da., 10,000 Da. to 80,000 Da., 20,000 Da. to 60,000 Da., and20,000 Da. to 40,000 Da. In a more preferred embodiment, the scaffold isapproximately the same mass as the therapeutic compound.

The invention provides a carrier-drug conjugate comprising a targetinggroup that is vitamin D, an analog, or a metabolite thereof that isnon-releasably conjugated to a therapeutic compound. In a preferredembodiment, the vitamin D is non-hormonal. In a more preferredembodiment, the non-hormonal vitamin D is not hydroxylated at the carbon1 position. In a more preferred embodiment, the therapeutic compound isconjugated at the carbon 3 position of the non-hormonal vitamin Dtargeting group. In a more preferred embodiment, the therapeuticcompound retains substantially the same activity as the therapeuticcompound not conjugated to the targeting group as measured by afunctional assay. In a more preferred embodiment, the targeting group isconjugated to the therapeutic peptide or said therapeutic nucleic acidvia a scaffold that is selected from the group consisting ofpoly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic compound. In a morepreferred embodiment, the scaffold is approximately the same mass as thetherapeutic compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(2k)-aldehyde adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an aldehyde coupling group.

FIG. 2: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(2k)-maleimide adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) a maleimide coupling group.

FIG. 3: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a Vitamin D-(3)-PEG_(1.3k)-NHS adduct. Thecarrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an NHS coupling group.

FIG. 4: Functional apelin assay measuring inhibition offorskolin-stimulated cAMP production in HEK293T cells expressing the APJreceptor. Apelin-13, Vitamin D-(25)-PEG_(2K)-C-apelin, and VitaminD-(3)-PEG_(2K)-apelin were tested. The functional activity of apelin(EC₅₀) was determined from a four parameter logistic function fit of thecurve.

FIG. 5: Pharmacokinetics of apelin and apelin conjugates. Apelin aloneor conjugated to the Vitamin D-(25)-PEG_(2k)-maleimide carrier, or theVitamin D-(3)-PEG_(2k)-aldehyde carrier were injected intravenously intoSprague-Dawley rats at 0.1 mg/kg. Plasma samples were analyzed forapelin concentration by ELISA in duplicate and the average value fromthree animals were plotted on the semi-log graph.

FIG. 6A: Improved pharmacokinetics of ghrelin conjugated to the VitaminD-(25)-PEG_(2k)-maleimide carrier and the VitaminD-(3)-PEG_(2k)-maleimide carrier when compared to unmodified ghrelin.Total (solid lines) and active (dashed lines) ghrelin were comparedfollowing intravenous injection into Sprague Dawley rats. FIG. 6B:Ghrelin conjugated to the Vitamin D-(3)-PEG_(2k)-maleimide carrier wascompared to unmodified ghrelin in rats following subcutaneous injectionsat t=0 and 48 hours and at various doses.

FIG. 7: Pharmacokinetics and bioavailability of total (solid lines) andactive (dashed lines) ghrelin and ghrelin conjugates delivered bysubcutaneous injection. Conjugation to the VitaminD-(25)-PEG_(2k)-maleimide carrier and the VitaminD-(3)-PEG_(2k)-maleimide carrier showed significant improvements overthe unconjugated ghrelin. The Vitamin D-(3)-PEG_(2k)-maleimide carrier,however, showed superior bioavailability and pharmacokinetic propertiescompared to the Vitamin D-(25)-PEG_(2k)-maleimide carrier.

FIG. 8: Pharmacokinetic profiles of intravenous (solid lines) andsubcutaneous (dashed lines) injections of ghrelin conjugated to PEG_(2k)alone or with Vitamin D-(25)-PEG_(2k)-maleimide carrier and the VitaminD-(3)-PEG_(2k)-maleimide carrier.

FIG. 9: Ghrelin and ghrelin conjugates of the VitaminD-(3)-PEG_(2k)-maleimide carrier treatment reverses body weight loss inrats bearing Yoshida AH130 ascites hepatoma cells as a model of cancercachexia.

FIG. 10: Vitamin D-(3)-PEG_(2k)-PTH and Vitamin D-(25)-PEG_(2k)-PTHpharmacokinetics were compared to unmodified PTH(1-34) upon subcutaneousinjection in rats.

FIG. 11: Vitamin D-(3)-PEG_(2k)-FGF21 and Vitamin D-(25)-PEG_(2k)-FGF21pharmacokinetics were compared to unmodified FGF21 upon subcutaneousinjection in rats.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides carrier-drug conjugates comprising targetinggroups that are non-hormonal vitamin D, vitamin D analogs, or vitamin Dmetabolites. Examples include vitamin D-based molecules that are nothydroxylated at the carbon 1 (C1) position. The carriers are linked totherapeutic compounds at the carbon 3 (C3) position. As disclosedherein, carrier groups are surprisingly effective when non-hormonalvitamin D forms are used and the therapeutic compound is linked to theCarbon 3 position. While not wishing to be bound by theory, it isbelieved that the hormonal forms of vitamin D are not appropriate forthe carriers described herein because they can be toxic due to theinduction of hypercalcemia. Also, because the hormonal forms bind thevitamin D receptor in cells, they may improperly target the carrier-drugconjugates to undesired cells or tissues. In contrast, non-hormonalvitamin D forms bind the Vitamin D Binding Protein (DBP) and remain incirculation longer.

The carrier molecules are attached to the therapeutic compounds usingchemistries described herein, described in WO2013172967, incorporatedherein in its entirety, or that are otherwise known in the art. Thecarriers improve the potency, absorption, bioavailability, circulatinghalf-life or pharmacokinetic properties of the therapeutic compounds. Incertain embodiments, the carriers further comprise what will bedescribed herein as a “scaffold” that acts, among other things, as anon-releasable “spacer” between the targeting group and the therapeuticcompound. In other embodiments, the carriers lack a scaffold.

The carriers are designed to be suitable for use in humans and animals.The carriers serve the purpose of improving the pharmacokineticproperties of a biological or chemical entity that is coupled,conjugated, or fused to the carrier. This occurs through the interactionof the targeting group with DBP. DBP can actively transport moleculesquickly and effectively from the site of administration to thecirculating plasma, thereby reducing exposure of the drug to degradativeenzymes. The carriers, by binding to DBP, also improve the circulatinghalf-life of the drug. This increases the potency and therapeuticefficacy of the drug by preventing kidney filtration and otherelimination processes.

The impact on patient health of this new class of therapies will beprofound. Many previously unusable therapies for serious conditions suchas ghrelin for cancer cachexia, apelin for pulmonary arterialhypertension, diabetes, and cardiac disease, and PTH forhypoparathyroidism could be realized by application of this invention.Improvements in other current therapies such as insulin and GLP1 fortreating diabetes could have a big impact on patient health andconvenience. A large number of diseases may benefit from RNAi-basedtreatments. These include diseases such as macular degeneration andHuntington's disease. Additionally, certain cancers, liver diseases, andinfectious diseases including respiratory syncytial virus, herpessimplex virus type 2, HIV, hepatitis A and B, influenza, and measles maybenefit from RNAi-based treatments.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

The term “absorption” is the movement of a drug into the bloodstream. Adrug needs to be introduced via some route of administration (e.g. oral,topical or dermal, subcutaneous, intramuscular, or intravenous) or in aspecific dosage form such as a tablet, patch, capsule or liquid.

An “antagonist” refers to a molecule capable of neutralizing, blocking,inhibiting, abrogating, reducing or interfering with the activities of aparticular or specified protein, including its binding to one or morereceptors in the case of a ligand, or binding to one or more ligands incase of a receptor. Antagonists include antibodies and antigen-bindingfragments thereof, proteins, peptides, glycoproteins, glycopeptides,glycolipids, polysaccharides, oligosaccharides, nucleic acids,bioorganic molecules, peptidomimetics, pharmacological agents and theirmetabolites, transcriptional and translation control sequences, and thelike. Antagonists also include small molecule inhibitors of proteins,hormones, or other bioactive molecules. Antagonists may be fusionproteins, receptor molecules, antisense molecules, aptamers, ribozymes,or derivatives that bind specifically to the proteins, hormones, orother bioactive molecules and thereby sequester its binding to itstarget.

“Antibodies” (Abs) and “immunoglobulins” (Igs) refer to glycoproteinshaving similar structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that generally lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Aptamers” are nucleic acid-based compounds that have been selected tobind a specific target. An example of an aptamer-based therapeuticcompound can be found in WO07/035922, incorporated by reference hereinin its entirety.

The term “bioavailability” refers to the fraction of an administereddose of unchanged drug that reaches the systemic circulation, one of theprincipal pharmacokinetic properties of drugs. When a medication isadministered intravenously, its bioavailability is 100%. When amedication is administered via other routes (such as orally), itsbioavailability generally decreases (due to incomplete absorption andfirst-pass metabolism) or may vary from patient to patient.Bioavailability is an important parameter in pharmacokinetics that isconsidered when calculating dosages for non-intravenous routes ofadministration.

“Carriers” are compounds that can be conjugated to, fused to, coupled toor formulated with therapeutic compounds to improve the absorption,half-life, bioavailability, pharmacokinetic or pharmacodynamicproperties of the drugs. They comprise a targeting group, a couplinggroup, and optionally, a scaffold moiety. In some embodiments, carriersmay carry a therapeutic compound from the site of subcutaneous injectioninto circulation as well as carry the therapeutic compound incirculation for an extended period of time.

An “effective amount” refers to an amount of therapeutic compound thatis effective, at dosages and for periods of time necessary, to achievethe desired therapeutic or prophylactic result. A “therapeuticallyeffective amount” of a therapeutic compound may vary according tofactors such as the disease state, age, sex, and weight of theindividual. A therapeutically effective amount may be measured, forexample, by improved survival rate, more rapid recovery, oramelioration, improvement or elimination of symptoms, or otheracceptable biomarkers or surrogate markers. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of thetherapeutic compound are outweighed by the therapeutically beneficialeffects. A “prophylactically effective amount” refers to an amount oftherapeutic compound that is effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,but not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

“Half-life” is a scientific term known in the art that refers to theamount of time that elapses when half of the quantity of a test moleculeis no longer detected. An in vivo half-life refers to the time elapsedwhen half of the test molecule is no longer detectable in circulatingserum or tissues of a human or animal.

A “hormone” is a biological or chemical messenger that communicatesbetween one cell (or group of cells) to another cell. As describedherein, hormones for use in the invention may be peptides, steroids,pheromones, interleukins, lymphokines, cytokines, or members of otherhormone classes known in the art.

“Homologs” are bioactive molecules that are similar to a referencemolecule at the nucleotide sequence, peptide sequence, functional, orstructural level. Homologs may include sequence derivatives that share acertain percent identity with the reference sequence. Thus, in oneembodiment, homologous or derivative sequences share at least a 70percent sequence identity. In a preferred embodiment, homologous orderivative sequences share at least an 80 or 85 percent sequenceidentity. In a more preferred embodiment, homologous or derivativesequences share at least an 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99 percent sequence identity. Homologous or derivativenucleic acid sequences may also be defined by their ability to remainbound to a reference nucleic acid sequence under high stringencyhybridization conditions. Homologs having a structural or functionalsimilarity to a reference molecule may be chemical derivatives of thereference molecule. Methods of detecting, generating, and screening forstructural and functional homologs as well as derivatives are known inthe art.

“Hybridization” generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel et al,Current Protocols in Molecular Biology, Wiley Interscience Publishers(1995).

An “individual,” “subject” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, primates (including human and non-human primates) androdents (e.g., mice, hamsters, guinea pigs, and rats). In certainembodiments, a mammal is a human. A “control subject” refers to ahealthy subject who has not been diagnosed as having a disease,dysfunction, or condition that has been identified in an individual,subject, or patient. A control subject does not suffer from any sign orsymptom associated with the disease, dysfunction, or condition.

A “medicament” is an active drug that has been manufactured for thetreatment of a disease, disorder, or condition.

“Morpholinos” are synthetic molecules that are non-natural variants ofnatural nucleic acids that utilize a phosphorodiamidate linkage,described in U.S. Pat. No. 8,076,476, incorporated by reference hereinin its entirety.

“Nucleic acids” are any of a group of macromolecules, either DNA, RNA,or variants thereof, that carry genetic information that may directcellular functions. Nucleic acids may have enzyme-like activity (forinstance ribozymes) or may be used to inhibit gene expression in asubject (for instance RNAi). The nucleic acids used in the inventionsdescribed herein may be single-stranded, double-stranded, linear orcircular. The inventions further incorporate the use of nucleic acidvariants including, but not limited to, aptamers, PNA, Morpholino, orother non-natural variants of nucleic acids. By way of example, nucleicacids useful for the invention are described in U.S. Pat. No. 8,076,476,incorporated by reference herein in its entirety.

“Patient response” or “response” can be assessed using any endpointindicating a benefit to the patient, including, without limitation, (1)inhibition, to some extent, of disease progression, includingstabilization, slowing down and complete arrest; (2) reduction in thenumber of disease episodes and/or symptoms; (3) inhibition (i.e.,reduction, slowing down or complete stopping) of a disease cellinfiltration into adjacent peripheral organs and/or tissues; (4)inhibition (i.e. reduction, slowing down or complete stopping) ofdisease spread; (5) decrease of an autoimmune condition; (6) favorablechange in the expression of a biomarker associated with the disorder;(7) relief, to some extent, of one or more symptoms associated with adisorder; (8) increase in the length of disease-free presentationfollowing treatment; or (9) decreased mortality at a given point of timefollowing treatment.

As used herein, the term “peptide” is any peptide comprising two or moreamino acids. The term peptide includes short peptides (e.g., peptidescomprising between 2-14 amino acids), medium length peptides (15-50) orlong chain peptides (e.g., proteins). The terms peptide, medium lengthpeptide and protein may be used interchangeably herein. As used herein,the term “peptide” is interpreted to mean a polymer composed of aminoacid residues, related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally-occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic peptides can besynthesized, for example, using an automated peptide synthesizer.Peptides can also be synthesized by other means such as by cells,bacteria, yeast or other living organisms. Peptides may contain aminoacids other than the 20 gene-encoded amino acids. Peptides include thosemodified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, and are well-known to those of skill in theart. Modifications occur anywhere in a peptide, including the peptidebackbone, the amino acid side chains, and the amino or carboxyl termini.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or nonaqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants such as ascorbic acid or glutathione, chelating agents, lowmolecular weight proteins or other stabilizers or excipients.

The term “pharmacokinetics” is defined as the time course of theabsorption, distribution, metabolism, and excretion of a therapeuticcompound. Improved “pharmacokinetic properties” are defined as:improving one or more of the pharmacokinetic properties as desired for aparticular therapeutic compound. Examples include but are not limitedto: reducing elimination through metabolism or secretion, increasingdrug absorption, increasing half-life, and/or increasingbioavailability.

“PNA” refers to peptide nucleic acids with a chemical structure similarto DNA or RNA. Peptide bonds are used to link the nucleotides ornucleosides together.

“Scaffolds” are molecules to which other molecules can be covalently ornon-covalently attached or formulated. The scaffolds of the inventionmay act as “spacers” between the targeting group and the drug. Spacersare molecular entities that provide physical distance between the twodistinct molecular entities. Scaffolds may also contain a reactive“linker” or may have beneficial therapeutic properties in addition tothe drug. Linkers are the sites of attachment from one molecular entityto another. Thus, the scaffolds of the invention may be, for example,PEG, serum albumin, thioredoxin, an immunoglobulin, a modifying groupthat contains a reactive linker, a water-soluble polymer, or atherapeutic compound. The scaffolds and linkers of the invention arestable (i.e. non-releasable). Non-releasable linkers have more stablechemical bonds than releasable linkers to allow the attached molecularentities to remain attached in vivo. In certain embodiments, however,they may be “releasable” under specific conditions. Releasable linkershave inherent instability and allow for the release of the attachedmolecules under certain conditions over time.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μl/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

The “therapeutic compounds” disclosed herein refer to small molecules,chemical entities, nucleic acids, nucleic acid derivatives, peptides,peptide derivatives, naturally-occurring proteins,non-naturally-occurring proteins, glycoproteins, and steroids that areadministered to subjects to treat diseases or dysfunctions or tootherwise affect the health of individuals. Non-limiting examples oftherapeutic compounds include polypeptides such as enzymes, hormones,cytokines, or antibody fragments, antibody derivatives, drugs thataffect metabolic function, as well as organic compounds such asanalgesics, antipyretics, anti-inflammatory agents, antibiotics,anti-viral compounds, anti-fungal compounds, cardiovascular drugs, drugsthat affect renal function, electrolyte metabolism, drugs that act onthe central nervous system, chemotherapeutic compounds, receptoragonists and receptor antagonists. Therapeutic compounds include, forexample, extracellular molecules such as serum factors including, butnot limited to, plasma proteins such as serum albumin, immunoglobulins,apolipoproteins or transferrin, or proteins found on the surface oferythrocytes or lymphocytes. Thus, exemplary therapeutic compoundsinclude small molecules, chemical entities, nucleic acids, nucleic acidderivatives, peptides, peptide derivatives, naturally-occurringproteins, non-naturally-occurring proteins, peptide-nucleic acids (PNA),stapled peptides, oligonucleotides, morpholinos, antisense drugs,RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones,cytokines, interferons, growth factors, blood coagulation factors,antibodies, antibody fragments, antibody derivatives, toxin-conjugatedantibodies, antibody-drug conjugates, metabolic effectors, analgesics,antipyretics, anti-inflammatory agents, antibiotics, anti-microbialagents, anti-viral agents, anti-fungal drugs, musculoskeletal drugs,cardiovascular drugs, renal drugs, pulmonary drugs, digestive diseasedrugs, hematologic drugs, urologic drugs, metabolism drugs, hepaticdrugs, neurological drugs, anti-diabetes drugs, anti-cancer drugs, drugsfor treating stomach conditions, drugs for treating colon conditions,drugs for treating skin conditions, and drugs for treating lymphaticconditions. The term “therapeutic compound” as used herein hasessentially the same meaning as the terms “drug” or “therapeutic agent.”

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed before or during the course of clinicalpathology. Desirable effects of treatment include preventing theoccurrence or recurrence of a disease or a condition or symptom thereof,alleviating a condition or symptom of the disease, diminishing anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, ameliorating or palliating the diseasestate, and achieving remission or improved prognosis. In someembodiments, methods and compositions of the invention are useful inattempts to delay development of a disease or disorder.

A “vitamin” is a recognized term in the art and is defined as afat-soluble or water-soluble organic substance essential in minuteamounts for normal growth and activity of the body and is obtainednaturally from plant and animal foods or supplements.

“Vitamin D” is a group of fat-soluble secosteroids. Several forms(vitamers) of vitamin D exist. The two major forms are vitamin D2 orergocalciferol, and vitamin D3 or cholecalciferol. Vitamin D without asubscript refers to vitamin D2, D3 or other forms known in the art. Inhumans, vitamin D can be ingested as cholecalciferol (vitamin D3) orergocalciferol (vitamin D2). Additionally, humans can synthesize it fromcholesterol when sun exposure is adequate. Cholecalciferol may bemodified in the liver or in vitro to 25-hydroxycholecalciferol(“25-hydroxy Vitamin D”). In the kidney or in vitro, 25-hydroxy vitaminD can be modified into the distinct hormonal form of 1, 25-hydroxyvitamin D.

“Vitamin D binding protein” or “DBP” is a naturally circulating serumprotein found in all mammals that, among other activities, can bind toand transport vitamin D and its analogs to sites in the liver and kidneywhere the vitamin is modified to its active form, and it retains vitaminD in its various forms in circulation for, on average, 30 days inhumans. A DBP protein sequence is disclosed in SEQ ID NO:14 and anexemplary nucleic acid sequence encoding the DBP protein sequence isdisclosed in SEQ ID NO:15. DBP has multiple naturally-occurringisoforms. Exemplary isoforms are available in the public sequencedatabases (e.g. Accession Nos. NM_001204306.1, NM_001204307.1,NM_000583.3, BC036003.1, M12654.1, X03178.1, AK223458, P_001191235.1,NP_000574.2, AAA61704.1, AAD13872.1, NP_001191236.1, AAA19662.2, 154269,P02774.1, EAX05645.1, AAH57228.1, AAA52173.1, AAB29423.1, AAD14249.1,AAD14250.1, and BAD97178.1).

The invention contemplates non-hormonal vitamin D conjugates that bindDBP or functional DBP variants and homologs that contain conservative ornon-conservative amino acid substitutions that substantially retain DBPactivity. DBP binding molecules or functional DBP variants may beidentified using known techniques and characterized using known methods(Bouillon et al., J Bone Miner Res. 6(10):1051-7 (1991), Teegarden et.al., Anal. Biochemistry 199(2):293-299 (1991), McLeod et al, J BiolChem. 264(2):1260-7 (1989), Revelle et al., J. Steroid Biochem.22:469-474 (1985)). The foregoing references are incorporated byreference herein in their entirety.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

The invention provides effective routes for administration of proteins,peptides, other biologics, nucleic acids, and small molecule drugs. Theinvention further provides effective routes of drug administration viatransdermal, oral, parenteral, subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, intracranial injection, infusion,inhalation, ocular, topical, rectal, nasal, buccal, sublingual, vaginal,or implanted reservoir modes.

In addition, the inventions described herein provide compositions andmethods for maintaining target binding activity, i.e. pharmacodynamics(PD), for therapeutic compounds. It further provides compositions andmethods for improving the pharmacokinetic (PK) profiles of therapeuticcompounds as described herein. The invention further providescompositions and methods for improved drug absorption profiles ascompared to the drug absorption profiles for the drugs using the sameroutes of administration or different routes of administration butwithout the inventions described herein. The invention further providescompositions and methods for improved drug bioavailability profiles ascompared to the drug bioavailability profiles for the drugs using thesame routes of administration or different routes of administration butwithout the carriers described herein. The invention further providescompositions and methods for improved drug half-life profiles ascompared to the drug half-life profiles for the drugs using the sameroutes of administration or different routes of administration butwithout the inventions described herein.

The invention also provides alternative routes of drug administrationthat are more cost-effective or favorable to the patients when comparedto the drugs without the inventions described herein.

The non-hormonal vitamin D carriers disclosed herein may improve theabsorption, half-life, bioavailability, or pharmacokinetic properties ofthe linked therapeutic compounds. While not wishing to be bound bytheory, the carriers have the properties of binding to the body'snatural DBP. DBP may transport the carrier-drug complex from the site ofadministration to the circulating serum. The vitamin D-DBP interactionmay retain the therapeutic compounds in circulation for an extendedperiod of time. This can prevent its excretion from the body andincrease the exposure of the therapeutic compound in the body to achievea longer lasting therapeutic effect. Additionally, a smaller dose ofdrug may be required when conjugated the carrier when compared to theunmodified form.

The therapeutic compound carrier conjugates of the invention typicallyhave about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting groupsindividually attached to a therapeutic compound. The structure of eachof the targeting groups attached to the therapeutic compound may be thesame or different. In preferred embodiments, one or more targetinggroups are stably or non-releasably attached to the therapeutic compoundat the N-terminus, C-terminus, or other portion of a therapeuticprotein. For example, a therapeutic compound carrier conjugate maycomprise a targeting group attached to the N-terminus and additionally atargeting group attached to a lysine residue. In another embodiment, atherapeutic compound carrier conjugate has a targeting group attached toa therapeutic protein via a modification such as a sugar residue as partof a glycosylation site, or on an acylation site of a peptide orattached to a phosphorylation site or other natural or non-naturalmodifications that are familiar to one skilled in the art. Alsocontemplated are attachment sites using a combination of sites mentionedabove. One preferred embodiment of the present invention comprises atargeting group that is attached to the therapeutic compound at onespecific site on a therapeutic compound. In another preferredembodiment, the attachment site on a protein may be a cysteine, lysine,the N-terminus or C-terminus.

In another embodiment, the scaffold is a pharmaceutically acceptablecarrier. In preferred embodiments, the scaffold is poly(ethyleneglycol), polylysine, polyethyleneimine, poly(propyleneglycol), apeptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, anucleic acid, a glycan, a modifying group that contain a reactivelinker, a water-soluble polymer, a small carbon chain linker, or anadditional therapeutic moiety.

In one embodiment, water-soluble scaffold moieties have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

Peptides can have mixed sequences or be composed of a single amino acid,e.g., poly(lysine). An exemplary polysaccharide is poly(sialic acid). Anexemplary poly(ether) is poly(ethylene glycol), e.g. m-PEG.□Poly(ethyleneimine) is an exemplary polyamine, and poly(acrylic) acidis a representative poly(carboxylic acid). The polymer backbone of thewater-soluble polymer can be poly(ethylene glycol) (i.e. PEG). However,it should be understood that other related polymers are also suitablefor use in the practice of this invention and that the use of the termPEG or poly(ethylene glycol) is intended to be inclusive and notexclusive in this respect. The term PEG includes poly(ethylene glycol)in any of its forms, including alkoxy PEG, difunctional PEG, multiarmedPEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein. The polymer backbone can belinear or branched.

Branched polymer backbones are generally known in the art. Typically, abranched polymer has a central branch core moiety and a plurality oflinear polymer chains linked to the central branch core. PEG is commonlyused in branched forms that can be prepared by addition of ethyleneoxide to various polyols, such as glycerol, pentaerythritol andsorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)m in which R represents thecore moiety, such as glycerol or pentaerythritol, and m represents thenumber of arms. Multi-armed PEG molecules, such as those described inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), polyvinylpyrrolidone), polylysine,polyethyleneimine, poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures thereof. Although themolecular weight of each chain of the polymer backbone can vary, it istypically in the range of about 100 Da to about 100,000 Da.

In other embodiments, the scaffold moiety may be a peptide, serumalbumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid,a glycan, a modifying group that contains a reactive linker, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic compound. In one embodiment, the scaffold moieties arenon-toxic to humans and animals. In another embodiment, the scaffoldsare endogenous serum proteins. In another embodiment, the scaffoldmoieties are water-soluble polymers. In another embodiment, thescaffolds are non-naturally-occurring polymers. In another embodiment,the scaffolds are naturally-occurring moieties that are modified bycovalent attachment to additional moieties (e.g., PEG, poly(propyleneglycol), poly(aspartate), biomolecules, therapeutic moieties, ordiagnostic moieties). The scaffolds and linkers of the invention arestable (i.e. non-releasable). In certain embodiments, however, they maybe “releasable” under specific conditions.

The conjugation of hydrophilic polymers, such as PEG, is known in theart. In its most common form, PEG is a linear polymer terminated at eachend with hydroxyl groups: HO—CH2CH2O—(CH2CH2O)n-CH2CH2-OH where ntypically ranges from about 3 to about 4000. In a preferred embodiment,the PEG has a molecular weight distribution that is essentiallyhomodisperse. In another preferred embodiment, the PEG is a linearpolymer. In another preferred embodiment the PEG is a branched polymer.

Many end-functionalized or branched derivatives and various sizes areknown in the art and commercially available. By way of example,conjugation of the PEG or PEO may be carried out using the compositionsand methods described herein and in U.S. Pat. No. 7,803,777 (Defrees etal.) and U.S. Pat. No. 4,179,337 (Davis et al.), each of which areincorporated by reference herein in their entirety.

In some embodiments, smaller therapeutic compounds are paired withsmaller scaffold moieties and larger therapeutic compounds are pairedwith larger scaffold moieties. It is contemplated, however, that smallertherapeutic compounds could be paired with a larger scaffold moiety andvice versa. Smaller therapeutic compounds are defined as having amolecular weight of 1 Da to 10 kDa. Larger therapeutic compounds aredefined as having a molecular weight of 10 kDa to 1000 kDa.

In some embodiments, a scaffold that is approximately equal to themolecular weight of a small therapeutic compound results in anefficacious carrier-drug conjugate. Improvements in efficacy may beobtained by empirically adjusting the scaffold size further. Withoutwishing to be bound by theory, the pharmacokinetic properties andefficacy of the conjugates may be enhanced when a scaffold (incombination with linkers as needed) is big enough to ablate potentialsteric hindrance of the drug by DBP binding and vice versa. Thus, atherapeutic compound is conjugated so that its active region is exposedand available for functional activity and the carrier is able to bindDBP. Additional embodiments provide non-releasable attachments thatextend the circulation of therapeutics. In some small peptideembodiments such as ghrelin, the scaffold may be selected to beapproximately equal to the molecular weight of the therapeutic. In somelarge protein embodiments, such as an antibody, the scaffold may be longenough to allow binding between the Vitamin D carrier and DBP.

In preferred embodiments, the conjugation of the therapeutic compoundretains substantially all of its activity following the conjugation. Theactive region of given therapeutic may be known in the art or determinedempirically. In other embodiments, the conjugate is therapeuticallyactive while remaining linked to the carrier. This embodiment maymaximize the time in circulation and as well as its efficacy.

The scaffolds of the present invention, for example, could have amolecular weight of 100 Daltons (Da.), 500 Da., 1000 Da., 2000 Da., 5000Da., 10,000 Da., 15,000 Da., 20,000 Da., 30,000 Da., 40,000 Da. or60,000 Da. In one embodiment of the invention, “small” scaffolds may bebetween about 100 Da. and 20,000 Da. In another embodiment, “large”scaffolds may be greater than about 20,000 Da. to about 200,000 Da. Inpreferred embodiments, the scaffold moiety is between about 100 Da. and200,000 Da. In more preferred embodiments, the scaffold is between about100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da. and 10,000 Da.,400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da. and 2,000 Da.,1000 Da. and 200,000 Da., 20.00 Da. and 200,000 Da., 100,000 and 200,000Da., 5000 Da. and 100,000 Da., 10,000 Da. and 80,000 Da., 20,000 Da. and60,000 Da., or 20,000 Da. and 40,000 Da. The size of the scaffolds maybe varied to maximize absorption, bioavailability, circulatinghalf-life, or efficacy of the conjugated therapeutic compound.

Another component of the carrier molecule preferably comprises acoupling group that is used to covalently attach the drug to thescaffold or the carrier. The coupling groups of the invention include anamine-reactive group, a thiol-reactive group, a maleimide group, a thiolgroup, an aldehyde group, an NETS-ester group, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido, combinations thereof, or other coupling groups familiarto persons skilled in the art. The coupling groups of the invention canpromote thiol linkages, amide linkages, oxime linkages, hydrazonelinkages, thiazolidinone linkages or utilize cycloaddition reactionsalso called click chemistry to couple the carrier to a therapeuticcompound. In another embodiment, the composition preferably includes acombination of one or more therapeutic compounds attached to thecoupling group of the scaffold molecule. The linkers of the inventionmay be between about 40 and 100 Daltons. In preferred embodiments, thelinkers may be between about 40-50, 50-60, 60-70, 70-80, 80-90, or90-100 Daltons. The linkers may also be varied to affect the stabilityor releasability of the link between the carrier and the therapeuticcompound.

NHS groups are known to those skilled in the art as being useful forcoupling to native peptides and proteins without having to engineer in asite of attachment. NHS groups allow attachment to most proteins andpeptides that contain amino acids with amine groups such as a lysineresidue. Utilization of NHS groups allows for flexibility in the site ofcarrier conjugation as protein structure and reaction time can influencethe attachment site and number of carrier molecules conjugated to thetherapeutic compound. By way of example, controlling the molar ratio ofNHS-carrier to therapeutic compound, one skilled in the art can havesome control over the number of carrier molecules attached to thetherapeutic compound thus allowing for more than one carrier to beconjugated to a given therapeutic compound, if desired.

Conjugation of the carrier to a therapeutic compound is achieved bymixing a solution of the molecules together in a specific molar ratiousing compatible solutions, buffers or solvents. For example, a molarratio of about 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1, 50:1, 100:1,1000:1, or about 1:2, 1:4, 1:5, 1:10, 1:20 1:25, 1:50, 1:100 or 1:1000of carrier to therapeutic compound could be used. By varying the ratio,this could result in different numbers of individual carriers attachedto the therapeutic compound, or could help to select a specific site ofattachment. Attachment of the carriers is also pH, buffer, salt andtemperature dependent and varying these parameters among otherparameters can influence the site of attachment, the number of carriersattached, and the speed of the reaction. For example, by selecting a pHfor the reaction at or below pH 6 could help selectively conjugate analdehyde version of the carrier to the N-terminus of the therapeuticprotein or peptide.

Additionally, in order to retain substantially the same activity of thetherapeutic compounds, conjugation to the carriers will be at a site onthe molecules that do not interfere with therapeutic function. Forproteins, it may require conjugation to the amino terminus, the carboxyterminus, or to an internal reactive amino acid. For nucleic acids, itmay require conjugation to the 5′ end, the 3′ end, or an internalnucleotide, nucleoside, or a derivative thereof. In one embodiment, thecarrier is conjugated to a nucleotide or nucleoside prior toincorporation into a polynucleotide molecule.

In certain embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, avitamin D-related metabolite, an analog of a vitamin Drelated-metabolite, a peptide that binds DBP, an anti-DBP antibody, ananti-DBP antibody derivative, a nucleotide aptamer that binds DBP, or asmall carbon-based molecule that binds DBP;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,polyethyleneimine, poly(propyleneglycol), a peptide, serum albumin,thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan,a modifying group that contains a reactive linker, polylactic acid, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic compound;C is an amine-reactive group, a thiol-reactive group, a maleimide group,a thiol group, a disulfide group, an aldehyde group, an NHS-ester group,a 4-nitrophenyl ester, an acylimidazole, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido or combinations thereof;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In preferred embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, avitamin D-related metabolite, an analog of a vitamin Drelated-metabolite, or a small carbon-based molecule that binds DBP;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine,poly(propyleneglycol), a peptide, serum albumin, an amino acid, anucleic acid, a glycan, polylactic acid, a water-soluble polymer, or asmall carbon chain linker;C is a maleimide group, a thiol group, a disulfide group, an aldehydegroup, an NETS-ester group, an iodoacetyl group, or a bromoacetyl group;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In more preferred embodiments, the present invention provides carriersthat include those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:B is a targeting group selected from vitamin D, a vitamin D analog, or avitamin D-related metabolite;S is a scaffold moiety, comprising poly(ethylene glycol), polylysine orpoly(propyleneglycol);C is a maleimide group, a disulfide group, an aldehyde group, anNHS-ester group or an iodoacetyl group;(L)^(a) and (M)^(b) are linkers independently selected from —(CH₂)_(n)—,—C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂—and —NH—;a is an integer from 0-4; andb is an integer from 0-4; andn is an integer from 0-3.

In most preferred embodiments, the present invention provides carriersthat include those of formulas IIa, IIb, and IIc:

Wherein:B is a targeting group selected from vitamin D, a vitamin D analog, or avitamin D-related metabolite;S is a scaffold moiety, comprising poly(ethylene glycol), orpoly(propyleneglycol); andC is a maleimide group, a disulfide group, an aldehyde group, anNHS-ester group or an iodoacetyl group;L¹ is —(CH₂)_(n)—;L³ is —(CH₂)_(o)—;(M)^(b) are linkers independently selected from —(CH₂)_(n)—, —C(O)NH—,—HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—, —S(O)₂— and —NH—;b is an integer from 0-4; andn is 3; ando is 1.

In PCT/US2013/031788, which is incorporated herein by reference,conjugation at the Carbon 25 (C25) position of 25-hydroxy-vitamin D₃ isexemplified. The present invention incorporates conjugation at the C3position of 25-hydroxy-vitamin D₃. This gives improved half-lifeextension and bioavailability compared to the C25 conjugates.

In certain most preferred embodiments of formula IIa, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVa.

In certain most preferred embodiments of formula IIb, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVb.

In certain most preferred embodiments of formula IIc, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVc.

In certain most preferred embodiment, S is between about 100 Da. and200,000 Da. In other most preferred embodiments, the scaffold moiety isbetween about 100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da.and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da.and 2,000 Da., 1000 Da. and 200,000 Da., 5000 Da. and 100,000 Da.,10,000 Da. and 80,000 Da., 20,000 Da. and 60,000 Da., or 20,000 Da. and40,000 Da.

In a specific embodiment, the present invention provides a carrierrepresented by formula V.

In another specific embodiment, the present invention provides a carrierrepresented by formula VI.

In another specific embodiment, the present invention provides a carrierrepresented by formula VII.

In certain embodiments, the present invention provides a method forproducing a carrier of formula I:B-(L)^(a)-S-(M)^(b)-C  Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Ib:HOOC-L³-S-(M)^(b)-C  Ibin the presence of an amide coupling agent,wherein B, S, C and L¹, L³, and (M)^(b) are defined as above and L² is—C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula I. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

One skilled in the art will recognize that any suitable leaving groupmay be coupled with the carboxylic acid of formula Ib in the presence ofa suitable coupling agent to form an active ester of formula Ic:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In some embodiments, thepresent invention provides a method for producing a carrier of formulaI:B-(L)^(a)-S-(M)^(b)-C  Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formulaROOC-L³-S-(M)^(b)-C  Icwherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIa:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Id:HOOC-L³-S-(M)^(b)-CH₂OH  Idin the presence of an amide coupling agent forming a compound of formulaIe; and

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, L¹, L³, (M)^(b), b, n and o are defined as above and L² is—C(O)NH— and C is an aldehyde group.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula Ie. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In certain embodiments, any suitable leaving group can be coupled with acarboxylic acid of formula Id in the presence of a suitable couplingreagent to form an active ester of formula If:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, the present invention provides a method forproducing a carrier of formula Ie:

comprising the step of reacting a compound of formula Ia;B-L¹-NH₂  Iawith a compound of formula If; andROOC-L³-S-(M)^(b)-CH₂OH  If

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIc:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Ig:ROOC—S-(M)^(b)-COOH  Igforming a compound of formula Ih; and

Converting a carboxylic acid of formula Ih to an active ester of formulaIIc;

wherein B, S, C, R, L¹, (M)^(b), b, n and o are defined as above and L²is —C(O)NH-·

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable leaving group can be coupled with a carboxylic acid offormula Ih in the presence of a suitable coupling reagent to form anactive ester of formula IIc. Suitable leaving groups include, but arenot limited to imidazole, HOBT, NHS and 4-nitrophenol. Suitable couplingreagents include, but are not limited to 2-chloromethylpyridiniumiodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a combination of a suitable leavinggroup and a coupling reagent.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to 1,1′-carbonyldiimidazole,N,N′-disuccinimidyl carbonate, 4-nitrophenyl trifluoroacetate and HBTU.In some embodiments, the single reagent is used alone. In otherembodiments, the single reagent is used with an acyl transfer catalyst.Such acyl transfer catalysts include, but are not limited to DMAP andpyridine. One skilled in the art will recognize that additional acyltransfer catalysts may be used.

In a specific embodiment, the present invention provides a method forproducing a carrier represented by formula V:

comprising the step of reacting a compound of formula Va:

with a compound of formula Vb:

to form a compound of formula Vc;

Reduction of the nitrile group to form the amine of formula Vd;

Reaction of the compound of formula Vd with a compound of formula Ve;

To form a compound of the formula Vf

Oxidation of the primary alcohol of formula Vf to form the aldehyde offormula V.

In some embodiments, the reaction of a compound of formula Vb with acompound of formula Va is promoted by addition of Triton B. One skilledin the art will recognize that other reagents may be used to promotenucleophilic addition to acrylonitrile.

In some embodiments, reduction of the nitrile of formula Vc to the amineof formula Vd is performed using AlCl₃/LAH. One skilled in the art willrecognize that other reduction reagents may be used including sodium,H₂/Pd, Hz/Raney nickel, and diborane.

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NETS-ester offormula Ve with the amine of formula Vd. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaV. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VI:

comprising the steps of reacting a compound of formula Vd:

in the presence of an amide coupling agent with a compound of formulaVIa:

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula VI. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VII:

comprising the steps of reacting a compound of formula Vd:

with a compound of formula VIIa:

forming a compound of formula VIIb; and

Converting a carboxylic acid of formula VIIb to an active ester offormula VII;

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NETS-ester offormula VIIa with the amine of formula Va. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

NHS can be coupled with a carboxylic acid of formula VIIb in thepresence of a suitable coupling reagent to form an active ester offormula VII. Suitable coupling reagents include, but are not limited to2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU, and T3P.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a combination of NHS and acoupling reagent.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to, N,N′-disuccinimidyl carbonate. In someembodiments, the single reagent is used alone. In other embodiments thereagent is used with an acyl transfer catalyst. Such acyl transfercatalysts include, but are not limited to DMAP and pyridine. One skilledin the art will recognize that additional acyl transfer catalysts may beused.

One skilled in the art will recognize that there are other methods toconjugate a linker and scaffold to the C3 position of vitamin Dderivatives and analogues. For example, the C3 hydroxy group may beacylated by various groups as practiced by N. Kobayashi, K. Ueda, J.Kitahori, and K. Shimada, Steroids, 57, 488-493 (1992); J. G Haddad, etal., Biochemistry, 31, 7174-7181 (1992); A. Kutner, R. P. Link, H. K.Schnoes, H. F. DeLuca, Bioorg. Chem., 14, 134-147 (1986); and R. Ray, S.A. Holick, N. Hanafin, and M. F. Holick, Biochemistry, 25, 4729-4733(1986). The foregoing references are incorporated by reference in theirentirety. One skilled in the art will recognize that these chemistriescould be modified to synthesize compounds of the formula I:B-(L)^(a)-S-(M)^(b)-C  Iwherein B, S, C, (L)^(a), and (M)^(b) are defined as above.

If desired, therapeutic compound carrier conjugates having differentmolecular weights can be isolated using gel filtration chromatographyand/or ion exchange chromatography. Gel filtration chromatography may beused to fractionate different therapeutic compound carrier conjugates(e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates onetargeting group molecule per therapeutic compound, “2-mer” indicates twotargeting groups attached to therapeutic compound, and so on) on thebasis of their differing molecular weights (where the differencecorresponds essentially to the average molecular weight of the targetinggroup).

Gel filtration columns suitable for carrying out this type of separationinclude Superdex and Sephadex columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, and (iii) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE).

Separation of therapeutic compound carrier conjugates can also becarried out by reverse phase chromatography using a reverse phase-highperformance liquid chromatography (RP-HPLC) C18 column (AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a DEAE- or CM-Sepharose ion exchange columnavailable from Amersham Biosciences. The resulting purified compositionsare preferably substantially free of the non-targeting group-conjugatedtherapeutic compound. In addition, the compositions preferably aresubstantially free of all other non-covalently attached targetinggroups.

As described herein, the carriers of the invention may be non-hormonal25-hydroxy vitamin D or analogs thereof having a coupling group on the3′ carbon. “25-hydroxy vitamin D analogs” as used herein includes bothnaturally-occurring vitamin D metabolite forms as well as otherchemically-modified forms. The carriers of the invention do not includean active (i.e. hormonal) form of vitamin D (typically having a hydroxylgroup at the 1 carbon). These compounds are based on the vitamin Dstructure and retain partial function of vitamin D (i.e. they interactwith DBP), albeit at varying affinities. The following list exemplifiesvitamin D analog forms known in the art. They may, however, be hormonalor have the C1 hydroxyl group. They are presented here solely for theirchemical properties as vitamin D analogs, not for their functionalhormonal properties: OCT, a chemically synthesized version of1,25(OH)2D3 with an oxygen atom at the 22 position in the side chain(Abe et. al., FEBS Lett. 226:58-62 (1987)); Gemini vitamin D analog,1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol(BXL0124) (So et al., Mol Pharmacol. 79(3):360-7 (2011)); Paricalcitol,a vitamin D2 derived sterol lacking the carbon-19 methylene group foundin all natural vitamin D metabolites (Slatopolsky et al., Am J. KidneyDis. 26: 852 (1995)); Doxercalciferol (1α-hydroxyvitamin D₂), likealfacalcidol (1α-hydroxyvitamin D₃), is a prodrug which is hydroxylatedin the liver to 1α,25(OH)₂D₂, however, unlike alfacalcidol,doxercalciferol is also 24-hydroxylated to produce 1α,24(S)—(OH)₂D₂(Knutson et al., Biochem Pharmacol 53: 829 (1997)); Dihydrotachysterols(DHT₂), hydroxylated in vivo to 25(OH)DHT₂, 1,25(OH)₂DHT₂ (McIntyre etal., Kidney Int. 55: 500 (1999)), ED-71, and eldecalcitol. See alsoErben and Musculoskel, Neuron Interact. 2(1):59-69 (2001) and Steddon etal. Nephrol. Dial. Transplant. 16 (10): 1965-1967 (2001). The foregoingreferences are incorporated by reference in their entirety.

In another embodiment, the carrier further comprises a pharmaceuticallyacceptable scaffold moiety covalently attached to the targeting groupand the therapeutic compound. The scaffold moiety of the carriers of theinvention does not necessarily participate in but may contribute to thefunction or improve the pharmacokinetic properties of the therapeuticcompound. The scaffolds of the invention do not substantially interferewith the binding of the targeting group to DBP. Likewise, the scaffoldsof the invention do not substantially interfere with structure orfunction of the therapeutic compound. The length of the scaffold moietyis dependent upon the character of the targeting group and thetherapeutic compound. One skilled in the art will recognize that variouscombinations of atoms provide for variable length molecules based uponknown distances between various bonds (Morrison, and Boyd, OrganicChemistry, 3rd Ed, Allyn and Bacon, Inc., Boston, Mass. (1977),incorporated herein by reference). Other scaffolds contemplated by theinvention include peptide linkers, protein linkers such as human serumalbumin or immunoglobulin family proteins or fragments thereof, nucleicacid linkers, small carbon chain linkers, carbon linkers with oxygen ornitrogen interspersed, or combinations thereof. In preferredembodiments, the linkers are non-releasable or stable.

Also within the scope of the invention are therapeutic peptides. Theterm peptide is meant to include a string of amino acids. The aminoacids in the peptides of the invention may be naturally-occurring ornon-naturally-occurring. The peptides of the invention may besynthesized chemically or biologically, and can include cysteine-richpeptides, circular peptides, stapled peptides, peptides that include D-or L-amino acids and mixtures thereof, peptidomimetics, peptide-nucleicacids (PNAs), and combinations thereof. Exemplary embodiments includeAIDS vaccines, allergy vaccines, anti-inflammatory peptides,anti-integrin peptides, anti-TCR vaccines, anti-allergy peptides,anti-cancer peptides, anti-fungal peptides, anti-bacterial peptides,anti-rheumatic peptides, anti-thrombin peptides, anti-viral peptides, GProtein-Coupled Receptor (GPCR) ligands and related peptides (e.g. thesecretin family), CGRP s, GPCR antagonists, CMV peptides, calpaininhibitors, collagenase inhibitors, DAP inhibitors, defensins, dialyticoligopeptides, Enhancins, endorphins, endothelin antagonists,fibronectin inhibitors, gastrin antagonists, ghrelin, glucagonantagonists, gonadorelin analogs, growth factor peptides, hypothalamichormones, pituitary hormones, peptides that control gut function andappetite, proinflammatory adipose tissue products, peptides thatstimulate stem cell proliferation, proinflammatory peptides, naturalproducts, herpes simplex vaccines, heparin binding peptides, hepatitis-Bvaccines, immunomodulating peptides, influenza vaccines, LHRHantagonists, opiod peptide derivatives, MMP inhibitors, MUC-1 vaccines,malaria vaccines, melanoma vaccines, meningitis vaccines, neuropeptides,opioid peptides, osteogenic growth peptides, osteoporosis peptides,papillomavirus vaccines, prostate cancer vaccines, RGD peptides, RSVvaccines, T cell receptor peptides and the like. The inventioncontemplates synthetic analogs thereof that would be improved asclinical products through further modification by the methods describedherein. Those skilled in the art will recognize many additionalcommercially important peptides that are amenable to modificationsdescribed herein to provide increased half-life, duration of action,absorption and/or bioavailability.

Also contemplated within the scope of embodiments described herein aretherapeutic peptides that are branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular peptides result frompost-translational natural processes and are also made by suitablesynthetic methods. In some embodiments, any peptide product describedherein comprises a peptide analog described above that is thencovalently attached to an alkyl-glycoside surfactant moiety.

Other embodiments include therapeutic peptide chains that are comprisedof natural and unnatural amino acids or analogs of natural amino acids.As used herein, peptide and/or protein “analogs” comprise non-naturalamino acids based on natural amino acids, such as tyrosine analogs,which includes para-substituted tyrosines, ortho-substituted tyrosines,and meta-substituted tyrosines, wherein the substituent on the tyrosinecomprises an acetyl group, a benzoyl group, an amino group, a hydrazine,an hydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C2-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like.

Additional embodiments include therapeutic peptide chains havingmodified amino acids. Examples include acylated amino acids at theε-position of Lysine, amino acids with fatty acids such as octanoic,decanoic, dodecanoic, tetradecanoic, hexadecanoic, octadecanoic,3-phenylpropanoic acids and the like, or with saturated or unsaturatedalkyl chains. (Zhang, L. and Bulaj, G (2012) Curr Med Chem 19:1602-1618, incorporated herein by reference in its entirety).

The invention further contemplates therapeutic peptide chains comprisingnatural and unnatural amino acids or analogs of natural amino acids. Insome embodiments, peptide or protein “analogs” comprise non-naturalamino acids based on natural amino acids, such as tyrosine analogs,which includes para-substituted tyrosines, ortho-substituted tyrosines,and meta-substituted tyrosines, wherein the substituent on the tyrosinecomprises an acetyl group, a benzoyl group, an amino group, a hydrazine,an hydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C2-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like. Examples of Tyr analogsinclude 2,4-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine,O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine and the like.Examples of lysine analogs include ornithine (Orn), homo-lysine,Ca-methyl-lysine (CMeLys), and the like. Examples of phenylalanineanalogs include, but are not limited to, meta-substitutedphenylalanines, wherein the substituent comprises a methoxy group, aC1-C20 alkyl group, for example a methyl group, an allyl group, anacetyl group, or the like. Specific examples include, but are notlimited to, 2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyl-tyrosine,3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)),3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic), fluorinated phenylalanines, isopropyl-phenylalanine,p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine,p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, andisopropyl-phenylalanine, and the like.

Also contemplated within the scope of embodiments are therapeuticpeptide chains containing nonstandard or unnatural amino acids known tothe art, for example, C-alpha-disubstituted amino acids such as Aib,Ca-diethylglycine (Deg), aminocyclopentane-1-carboxylic acid (Ac4c),aminocyclopentane-1-carboxylic acid (Ac5c), and the like. Such aminoacids frequently lead to a restrained structure, often biased toward analpha helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem7: 105-117, incorporated herein by reference in its entirety).Additional examples of such unnatural amino acids useful in analogdesign are homo-arginine (Har) and the like. Substitution of reducedamide bonds in certain instances leads to improved protection fromenzymatic destruction or alters receptor binding. By way of example,incorporation of a Tic-Phe dipeptide unit with a reduced amide bondbetween the residues (designated as Tic-F[CH2-NH]^-Phe) reducesenzymatic degradation.

In some embodiments, modifications at the amino or carboxyl terminus mayoptionally be introduced into the present peptides or proteins (Nestor,J. J., Jr. (2009) Current Medicinal Chemistry 16: 4399-4418). Forexample, the present peptides or proteins can be truncated or acylatedon the N-terminus (Gourlet, P., et al. (1998) Eur J Pharmacol 354:105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9: 483-494), thecontents of which is incorporated herein by reference in theirentirety). Other modifications to the N-terminus of peptides orproteins, such as deletions or incorporation of D-amino acids such asD-Phe result in potent and long acting agonists or antagonists whensubstituted with the modifications described herein such as long chainalkyl glycosides.

Thus, the invention provides therapeutic compound analogs wherein thenative therapeutic compound is modified by acetylation, acylation,PEGylation, ADP-ribosylation, amidation, covalent attachment of a lipidor lipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-link formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, (Nestor, J. J., Jr. (2007)Comprehensive Medicinal Chemistry II 2: 573-601, Nestor, J. J., Jr.(2009) Current Medicinal Chemistry 16: 4399-4418, Uy, R. and Wold, F.(1977) Science 198:890-6, Seifter, S. and Englard, S. (1990) MethodsEnzymol 182: 626-646, Rattan, S. I., et al. (1992) Ann NY Acad Sci 663:48-62). The foregoing references are incorporated by reference in theirentirety.

Glycosylated therapeutic peptides may be prepared using conventionalFmoc chemistry and solid phase peptide synthesis techniques, e.g., onresin, where the desired protected glycoamino acids are prepared priorto peptide synthesis and then introduced into the peptide chain at thedesired position during peptide synthesis. Thus, the therapeutic peptidepolymer conjugates may be conjugated in vitro. The glycosylation mayoccur before deprotection. Preparation of amino acid glycosides isdescribed in U.S. Pat. No. 5,767,254, WO 2005/097158, and Doores, K., etal., Chem. Commun., 1401-1403, 2006, which are incorporated herein byreference in their entirety. For example, alpha and beta selectiveglycosylations of serine and threonine residues are carried out usingthe Koenigs-Knorr reaction and Lemieux's in situ anomerizationmethodology with Schiff base intermediates. Deprotection of the Schiffbase glycoside is then carried out using mildly acidic conditions orhydrogenolysis. A composition, comprising a glycosylated therapeuticpeptide conjugate is made by stepwise solid phase peptide synthesisinvolving contacting a growing peptide chain with protected amino acidsin a stepwise manner, wherein at least one of the protected amino acidsis glycosylated, followed by water-soluble polymer conjugation. Suchcompositions may have a purity of at least 95%, at least 97%, or atleast 98%, of a single species of the glycosylated and conjugatedtherapeutic peptide.

Monosaccharides that may by used for introduction at one or more aminoacid residues of the therapeutic peptides defined and/or disclosedherein include glucose (dextrose), fructose, galactose, and ribose.Additional monosaccharides suitable for use include glyceraldehydes,dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose,xylose, ribulose, xylulose, allose, altrose, mannose, N-Acetylneuraminicacid, fucose, N-Acetylgalactosamine, and N-Acetylglucosamine, as well asothers. Glycosides, such as mono-, di-, and trisaccharides for use inmodifying a therapeutic peptide, one or more amino acid residues of thetherapeutic peptides defined and/or disclosed herein include sucrose,lactose, maltose, trehalose, melibiose, and cellobiose, among others.Trisaccharides include acarbose, raffinose, and melezitose.

In further embodiments of the invention, the therapeutic compoundsdefined and/or disclosed herein may be chemically coupled to biotin. Thebiotin/therapeutic compound can then bind to avidin.

Also within the scope of the invention are polypeptides that areantibodies. The term antibody is meant to include monoclonal antibodies,polyclonal antibodies, toxin-conjugated antibodies, drug-conjugatedantibodies (ADCs), humanized antibodies, antibody fragments (e.g., Fcdomains), Fab fragments, single chain antibodies, bi- or multi-specificantibodies, Llama antibodies, nano-bodies, diabodies, affibodies, Fv,Fab, F(ab′)2, Fab′, scFv, scFv-Fc, and the like. Also included in theterm are antibody-fusion proteins, such as Ig chimeras. Preferredantibodies include humanized or fully human monoclonal antibodies orfragments thereof.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, monovalentantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity) and may also include certain antibody fragments (as describedin greater detail herein). An antibody can be chimeric, human, humanizedand/or affinity matured.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containthe Fc region. “Antibody fragments” comprise a portion of an intactantibody, preferably comprising the antigen binding region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies.

In certain embodiments, such a monoclonal antibody typically includes anantibody comprising a polypeptide sequence that binds a target, whereinthe target-binding polypeptide sequence was obtained by a process thatincludes the selection of a single target binding polypeptide sequencefrom a plurality of polypeptide sequences. For example, the selectionprocess can be the selection of a unique clone from a plurality ofclones, such as a pool of hybridoma clones, phage clones, or recombinantDNA clones. It should be understood that a selected target bindingsequence can be further altered, for example, to improve affinity forthe target, to humanize the target binding sequence, to improve itsproduction in cell culture, to reduce its immunogenicity in vivo, tocreate a multispecific antibody, etc., and that an antibody comprisingthe altered target binding sequence is also a monoclonal antibody ofthis invention. In contrast to polyclonal antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. In addition to their specificity, monoclonal antibodypreparations are advantageous in that they are typically uncontaminatedby other immunoglobulins.

Antibodies that bind specifically to an antigen have a high affinity forthat antigen. Antibody affinities may be measured by a dissociationconstant (Kd). In certain embodiments, an antibody provided herein has adissociation constant (Kd) of equal to or less than about 100 nM, 10 nM,1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g. 10⁻⁷M or less, from 10⁻⁷ M to10⁻¹³M, from 10⁻⁸M to 10⁻¹³ M or from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (125I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [125I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with, e.g., immobilized antigen CM5chips at ^(˜)10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NETS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml(^(˜)0.2 μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (K_(on)) and dissociation rates (K_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1s-1 by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette. Other coupling chemistries for the target antigen tothe chip surface (e.g., streptavidin/biotin, hydrophobic interaction, ordisulfide chemistry) are also readily available instead of the aminecoupling methodology (CM5 chip) described above, as will be understoodby one of ordinary skill in the art.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler et al, Nature, 256: 495 (1975); Harlow et al,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas pp. 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567), phage display technologies (see,e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J.Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132(2004), andtechnologies for producing human or human-like antibodies in animalsthat have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO98/24893;WO96/34096; WO96/33735; WO91/10741; Jakobovits et al., Proc. Natl. Acad.Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Markset al., Bio. Technology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995). The above patents, publications, and references are incorporatedby reference in their entirety.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or nonhuman primate having the desired specificity,affinity, and/or capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994). The foregoing references are incorporated by referencein their entirety.

A “human antibody” is one which comprises an amino acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. Such techniques include screening human-derivedcombinatorial libraries, such as phage display libraries (see, e.g.,Marks et al., J. Mol. Biol, 222: 581-597 (1991) and Hoogenboom et al.,Nucl. Acids Res., 19: 4133-4137 (1991)); using human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies (see, e.g., Kozbor, J. Immunol, 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 55-93 (Marcel Dekker, Inc., New York, 1987); andBoerner et al., J. Immunol, 147: 86 (1991)); and generating monoclonalantibodies in transgenic animals (e.g., mice) that are capable ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production (see, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature,362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993)).This definition of a human antibody specifically excludes a humanizedantibody comprising antigen-binding residues from a non-human animal.

All known types of such antibodies are within the scope of theinvention. Exemplary antibodies include those that bind to growthfactors, cytokines, lymphokines, cell surface receptors, enzymes,vascular endothelial growth factors, fibroblast growth factors, andantibodies to their respective receptors. Other exemplary antibodiesinclude monoclonal antibodies directed to receptor-IgG Fc fusionproteins, and glycoproteins. Any modified (e.g., mutated) version of anyof the above listed polypeptides is also within the scope of theinvention. Therapeutic compounds to be used in the invention are knownin the art and are disclosed by way of example in U.S. Pat. No.7,608,681, incorporated herein by reference in its entirety.Additionally, the invention contemplates conjugates of inhibitors orantagonists of naturally-occurring or non-naturally occurring antibodiesin a subject that cause autoimmune diseases or undesirable inflammatoryconditions.

In one embodiment, the drug is a DNA molecule, an RNA molecule, anaptamer (single-stranded or double-stranded), DNA or RNAoligonucleotides, larger DNA molecules that are linear or circular,oligonucleotides that are used for RNA interference (RNAi), variationsof DNA such as substitution of DNA/RNA hybrid molecules, syntheticDNA-like molecules such as PNA or other nucleic acid derivativemolecules (see WO07/035922, incorporated by reference herein in itsentirety). In another embodiment, the therapeutic compound is composedof nuclease-resistant DNA or RNA oligonucleotides. In a preferredembodiment, nuclease-resistant DNA oligonucleotides are Morpholinos,(i.e. phosphorodiamidate analogs of nucleic acids that bind to nucleicacids in a sequence-specific manner, Sarepta Therapeutics, CambridgeMass.).

In other embodiments, RNAi conjugated to the vitamin D carriers of theinvention are used to treat both inherited and infectious diseases. Inpreferred embodiments, the conjugates are used to treat, for example,blood conditions, liver conditions, cardiovascular conditions,hepatitis, eye conditions, metabolic conditions, graft rejections,cancer, autoimmune conditions, amyloidosis, and nervous systemconditions.

In another embodiment, the drug is a small molecule or chemical entity.In another embodiment, the drug is a peptide or a derivative of apeptide such as a PNA. In another embodiment, the drug is a proteincomprised of all or part of a polypeptide, whether full-length or afragment or truncated version, whether PEGylated, glycosylated orotherwise covalently or noncovalently modified or left unmodified.

Some aspects of the assembly of carriers utilizes chemical methods thatare well-known in the art. For example, Vitamin E-PEG is manufactured byEastman Chemical, Biotin-PEG is manufactured by many PEG manufacturerssuch as Enzon, Nektar and NOF Corporation. Methods of producing PEGmolecules with some vitamins and other therapeutic compounds linked tothem follow these and other chemical methods known in the art. Theattachment of PEG to an oligonucleotide or related molecule occurs, forexample, as the PEG2-N-hydroxysuccinimide ester coupled to theoligonucleotide through the 5′ amine moiety. Several coupling methodsare contemplated and include, for example, NHS coupling to amine groupssuch as a lysine residue on a peptide, maleimide coupling to sulfhydrylgroup such as on a cysteine residue, iodoacetyl coupling to a sulfhydrylgroup, pyridyldithiol coupling to a sulfhydryl group, hydrazide forcoupling to a carbohydrate group, aldehyde for coupling to theN-terminus, or tetrafluorophenyl ester coupling that is known to reactwith primary or secondary amines. Other possible chemical couplingmethods are known to those skilled in the art and can be substituted. Byway of example, conjugation using the coupling groups of the inventionmay be carried out using the compositions and methods described inWO93/012145 (Atassi et al.) and also see U.S. Pat. No. 7,803,777(Defrees et al.), incorporated by reference herein in their entirety.

Exemplary drug formulations of the invention include aqueous solutions,organic solutions, powder formulations, solid formulations and a mixedphase formulations.

Pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, and pharmaceutically acceptablesalts thereof, with any pharmaceutically acceptable carrier, adjuvant orvehicle. Pharmaceutically acceptable carriers, adjuvants and vehiclesthat may be used in the pharmaceutical compositions of this inventioninclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium tri silicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Pharmaceutically acceptable salts retain the desired biological activityof the therapeutic composition without toxic side effects. Examples ofsuch salts are (a) acid addition salts formed with inorganic acids, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like/and salts formed with organic acids suchas, for example, acetic acid, trifluoroacetic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tanic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid and the like; (b) base additionsalts or complexes formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium, and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethlenediamine; or (c) combinations of(a) and (b), e.g. a zinc tannate salt and the like.

The pharmaceutical compositions of this invention may be administered bysubcutaneous, transdermal, oral, parenteral, inhalation, ocular,topical, rectal, nasal, buccal (including sublingual), vaginal, orimplanted reservoir modes. The pharmaceutical compositions of thisinvention may contain any conventional, non-toxic,pharmaceutically-acceptable carriers, adjuvants or vehicles. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, and intracranial injection or infusiontechniques.

Also contemplated, in some embodiments, are pharmaceutical compositionscomprising as an active ingredient, therapeutic compounds describedherein, or pharmaceutically acceptable salt thereof, in a mixture with apharmaceutically acceptable, non-toxic component. As mentioned above,such compositions may be prepared for parenteral administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops, evaporating solutions or aerosols; for inhalation,particularly in the form of liquid solutions or dry powders withexcipients, defined broadly; for transdermal administration,particularly in the form of a skin patch or microneedle patch; and forrectal or vaginal administration, particularly in the form of asuppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example, as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.(1985), incorporated herein by reference in its entirety. Formulationsfor parenteral administration may contain as excipients sterile water orsaline alkylene glycols such as propylene glycol, polyalkylene glycolssuch as polyethylene glycol, saccharides, oils of vegetable origin,hydrogenated napthalenes, serum albumin or other nanoparticles (as usedin Abraxane™, American Pharmaceutical Partners, Inc. Schaumburg, Ill.),and the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid or solutions in evaporating solvents such ashydrofluorocarbons, and may contain excipients for stabilization, forexample, saccharides, surfactants, submicron anhydrous alpha-lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration, typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

Delivery of modified therapeutic compounds described herein to a subjectover prolonged periods of time, for example, for periods of one week toone year, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, polymeric hydrogels,osmotic pumps, vesicles, micelles, liposomes, transdermal patches,iontophoretic devices and alternative injectable dosage forms may beutilized for this purpose. Localization at the site to which delivery ofthe active ingredient is desired is an additional feature of somecontrolled release devices, which may prove beneficial in the treatmentof certain disorders.

In certain embodiments for transdermal administration, delivery acrossthe barrier of the skin would be enhanced using electrodes (e.g.iontophoresis), electroporation, or the application of short,high-voltage electrical pulses to the skin, radiofrequencies, ultrasound(e.g. sonophoresis), microprojections (e.g. microneedles), jetinjectors, thermal ablation, magnetophoresis, lasers, velocity, orphotomechanical waves. The drug can be included in single-layerdrug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, orvapor style patches, or could utilize patchless technology. Deliveryacross the barrier of the skin could also be enhanced usingencapsulation, a skin lipid fluidizer, or a hollow or solidmicrostructured transdermal system (MTS, such as that manufactured by3M), jet injectors. Additives to the formulation to aid in the passageof therapeutic compounds through the skin include prodrugs, chemicals,surfactants, cell penetrating peptides, permeation enhancers,encapsulation technologies, enzymes, enzyme inhibitors, gels,nanoparticles and peptide or protein chaperones.

One form of controlled-release formulation contains the therapeuticcompound or its salt dispersed or encapsulated in a slowly degrading,non-toxic, non-antigenic polymer such as copoly(lactic/glycolic) acid,as described in the pioneering work of Kent et al., U.S. Pat. No.4,675,189, incorporated by reference herein. The compounds, or theirsalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J R Robinson ed., Marcel Dekker Inc., New York, 1978;and Controlled Release of Biologically Active Agents, R W Baker, JohnWiley & Sons, New York, 1987. The foregoing are incorporated byreference in their entirety.

An additional form of controlled-release formulation comprises asolution of biodegradable polymer, such as copoly(lactic/glycolic acid)or block copolymers of lactic acid and PEG, is a bioacceptable solvent,which is injected subcutaneously or intramuscularly to achieve a depotformulation. Mixing of the therapeutic compounds described herein withsuch a polymeric formulation is suitable to achieve very long durationof action formulations.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be further enhanced by surfactants, such as,for example, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehdryocholic acid,glycodeoxycholic acid, cycledextrins and the like in an amount in therange of between about 0.1 and 15 weight percent, between about 0.5 and4 weight percent, or about 2 weight percent. An additional class ofabsorption enhancers reported to exhibit greater efficacy with decreasedirritation is the class of alkyl maltosides, such as tetradecylmaltoside(Arnold, J J et al., 2004, J Pharm Sci 93: 2205-13; Ahsan, F et al.,2001, Pharm Res 18:1742-46) and references therein, all of which arehereby incorporated by reference.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as Ph. Helv or a similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient that is solid at roomtemperature but liquid at the rectal temperature and therefore will meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topical transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the therapeutic compound to readilydispersed solids such as diketopiperazines (for example, Technosphereparticles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv2:1097-1106) or similar structures gives a formulation that results inrapid initial uptake of the therapeutic compound. Lyophilized powders,especially glassy particles, containing the therapeutic compound and anexcipient are useful for delivery to the lung with good bioavailability,for example, see Exubera® (inhaled insulin, Pfizer, Inc. and AventisPharmaceuticals Inc.) and Afrezza® (inhaled insulin, Mannkind, Corp.).

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably 0.5 and about 50 mg/kg body weight per day of the activeingredient compound are useful in the prevention and treatment ofdisease. Such administration can be used as a chronic or acute therapy.The amount of drug that may be combined with the carrier to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. A typical preparation will containfrom about 5% to about 95% active compound (w/w). Preferably, suchpreparations contain from about 20% to about 80% active compound.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, gender, diet, time of administration,rate of excretion, drug combination, the severity and course of aninfection, the patient's disposition to the infection and the judgmentof the treating physician.

The carrier-drug conjugates described herein provide advantages to drugmanufacturers and patients over unmodified drugs. Specifically, thecarrier-drug conjugate or formulation will be a more potent, longerlasting, and require smaller and less frequent dosing. This translatesinto lowered healthcare costs and more convenient drug administrationschedules for patients. The carrier-drug conjugates can also providesubcutaneous or transdermal routes of administration as alternatives tointravenous injection. These routes can be self-administered by patientsand thus improve patient compliance.

In yet another aspect of the invention, the levels of DBP can beincreased as part of the carrier-drug therapy. It has been reported thatestrogen can increase DBP levels (Speeckaert et al., Clinica ChimicaActa 371:33). It is contemplated here that levels of DBP can beincreased by administration of estrogen for more effective delivery ofcarrier-drug conjugates.

In yet another aspect of the invention, it is contemplated that thecarrier can be used to deliver drugs transdermally. Since DBP normallytransports UV activated vitamin D at locations close to the surface ofthe skin, the use of a transdermal delivery system with the carrierbecomes feasible.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner. Inparticular, the compositions and methods disclosed herein function withall non-hormonal forms of vitamin D, including homologs, analogs, andmetabolites thereof. This includes vitamin D as used in the examplesbelow.

EXAMPLES Example 1 Preparation Exemplary Carriers for CouplingTherapeutic Compounds to Non-Hormonal Vitamin D at the C25 Position

Exemplary carriers were prepared containing vitamin D and 2 kDa PEGscaffolds. One exemplary carrier was thiol-reactive and comprisedvitamin D-PEG with a maleimide reactive group at the C25 position(herein referred to as Vitamin D-(25)-PEG_(2k)-maleimide orVitD-(25)-PEG_(2k)-maleimide).

Another exemplary carrier was amine-reactive and comprised vitamin D-PEGwith an NETS-reactive group. These reagents were prepared as describedin WO2013172967 (Soliman et al.), incorporated herein by reference inits entirety.

Example 2 Preparation of an Exemplary Amino-Terminal Reactive Carrierfor Coupling Therapeutic Compounds to Non-Hormonal Vitamin D at the C3Position

An exemplary amino-terminal reactive carrier was prepared containing analdehyde reactive group connected to the C3 position of vitamin D and a2 kDa PEG scaffold (herein referred to as VitaminD-(3)-PEG_(2k)-aldehyde or VitD-(3)-PEG_(2k)-maleimide). The aldehyde onthe carrier in this example was used to conjugate to a freeamino-terminus on the proteins and peptides disclosed in the examplesbelow. The synthesis is outlined in FIG. 1.

Briefly,(S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol(compound Va, 20 mg, 0.049 mmol, 1 equiv., purchased from TorontoResearch Chemicals, catalog number C125700, also known as calcifedioland 25-hydroxyvitamin D) was dissolved in a mixture of anhydroustert-butanol and acetonitrile (10:1, 1 mL), cooled to 4° C.Acrylonitrile (26.6 mg, 0.5 mmol, 10 equiv.) was added to it followed byTriton B, 40% aqueous solution, 10 μL). The mixture was stirred at 4° C.for 2.5 h. The reaction was quenched with cold 2% HCl (10 mL), theaqueous phase was extracted with ether (2×10 mL), dried (MgSO₄) andevaporated to obtain the crude product. This material was purified byflash chromatography (TLC, silica gel, 50% ethyl acetate in hexanes)with 5-20% EtOAc/hexanes as eluent to isolate the desired product,3-(((S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)propanenitrile,compound V (15 mg, 68%) as a white solid (R_(f) 0.2 silica gel, 40%EtOAc in hexanes). NMR analysis did not show any appreciable amount ofsolvents.

To a solution of aluminum chloride (66 mg, 0.495 mmol) in anhydrousether (2 mL) at 0° C. under argon was added a solution of lithiumaluminum hydride (1M in ether, 19 mg, 0.5 mL, 0.5 mmol) dropwise. Themixture was stirred for 5 min., a solution of compound Vc (15 mg, 0.033mmol) in ether (3 mL) was added to it dropwise, the reaction mixture wasstirred at 0° C. for 5 min and then at room temperature for 1 h. Thereaction was monitored by MS and TLC (silica gel, 10% MeOH/CHCl₃/0.1%NH₄OH). Ethyl acetate (1 mL) and water (1 mL) were added to the reactionmixture followed by 5% NaOH (5 mL). The organic phase was separated, andthe aqueous phase was extracted with ethyl acetate (5 mL) and ether (5mL). The combined organic phases were washed with brine (5 mL), dried(Na₂SO₄) and evaporated on a rotavap to afford the desired amine,(R)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-(3-aminopropoxy)-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylheptan-2-ol,compound Vd (12.5 mg, 82%) as a pale yellow oil. R_(f) 0.2 (silica gel,20% MeOH/DCM/0.2% NH₄OH). The NMR analysis revealed the presence ˜8% ofethyl acetate.

Compound Vd (12.5 mg, 0.0273 mmol, 1 equiv.), compound Ve (hydroxyl PEGNHS ester, MW 2000 with n≅45 where n is the number of repeating CH₂CH₂Ounits, Jenkem Technology USA #A-5076, 43 mg, 0.0216 mmol, 0.8 equiv.)were dissolved in anhydrous dichloromethane (0.1 mL). Triethylamine (12mg, 16 μl mmol, 4 equiv.) was added and the reaction mixture was stirredfor 20 h at room temperature under nitrogen. The sample was dried undera stream of nitrogen to afford the crude compound Vf, which was purifiedby flash chromatography using 5-10% MeOH/dichloromethane as eluent toisolate the desired product Vf as a white foam (30 mg, 38%). R_(f) 0.4(silica gel, 10% methanol in dichloromethane). ¹H NMR analysis of theisolated material confirmed its identity and purity.

To a solution of compound Vf (30 mg, 0.0123 mmol, 1 equiv.),tetrapropylammonium perruthenate (1.0 mg, 0.00284, 0.23 equiv.) andN-methylmorpholine-N-Oxide (4.3 mg, 0.0369 mmol, 3equiv.) in 2 mL of drydichloromethane was added powdered 4 A° molecular sieves (500 mg) andthe reaction mixture was flushed with N₂. The reaction flask was coveredwith aluminum foil to avoid light and it was stirred at room temperaturefor 36 h. Since the R_(f) of both starting material and product is sameon TLC (silicagel, 10% MeOH/dichloromethane), formation of the productwas confirmed by examining the ¹H NMR of an aliquot. The reactionmixture was filtered through the pad of Celite in a pipette withdichloromethane (15 mL) and N₂ pressure. The combined organics wereconcentrated under a flow of N₂ and dried on high vacuum for 2 h to get35 mg (100%) of the crude product TLC (R_(f): 0.3, 10%MeOH/dichloromethane, staining with PMA). A second run of reaction underthe exactly same conditions yielded another 35 mg of the product. ¹H NMRof the product from both batches is same and hence combined to get 70 mgof compound V, VitD-(3)-PEG_(2k)-aldehyde.

Example 3 Preparation of an Exemplary Thiol-Reactive Carrier forCoupling Therapeutic Compounds to Non-Hormonal Vitamin D at the C3Position

An exemplary thiol-reactive carrier comprising vitamin D with amaleimide reactive group connected to the C3 position of vitamin D(VitD-(3)-PEG_(2k)-maleimide) was prepared. The maleimide on the carrierin this example was used to conjugate to a free thiol on the protein andpeptide in the examples below. The synthesis is outlined in FIG. 2.

Briefly, compound Vd (23 mg, 0.05 mmol, 1 equiv.) prepared as in Example2, compound VIa (Creative Pegworks cat. # PHB-956, MAL-PEG-COOH, 2 kwith n≅45 where n is the number of repeating CH₂CH₂O units, 79 mg,0.0395 mmol, 0.8 equiv.) and 2-chloro-1-methylpyridinium iodide (32 mg,0.125 mmol, 2.5 equiv.) were dissolved in anhydrous dichloromethane (1mL). Triethylamine (20.4 mg, 28 μl, 0.2 mmol, 4 equiv.) was added andthe reaction mixture was stirred for 4 h at room temperature undernitrogen. The reaction mixture was diluted with dichloromethane (20 mL),washed with 5% aqueous citric acid (20 mL), saturated aqueous sodiumbicarbonate (20 mL), and brine (20 mL). The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated at 30° C. The samplewas purified by silica gel (10 g) flash chromatography. The column waseluted with 1-10% MeOH/dichloromethane. Fractions containing pureproduct were combined together and evaporated on a rotavap, whilemaintaining the temperature at 30° C. The sample was dried under astream of nitrogen to afford compound VI, VitD-(3)-PEG_(2k)-maleimide asa brown gum (58 mg, 48%) (R_(f) 0.25, silica gel, 10% methanol indichloromethane). ¹H NMR analysis of the isolated material confirmed itsidentity and purity.

Example 4 Preparation of an Exemplary Amine-Reactive Carrier forCoupling Therapeutic Compounds to Non-Hormonal Vitamin D at the C3Position

An exemplary amine-reactive carrier comprising vitamin D with an NHSreactive group connected to the C3 position of vitamin D (Hereinreferred to as Vitamin D-(3)-PEG_(1.3k)-NHS or VitD-(3)-PEG_(1.3k)-NHS)was prepared. The NHS on the carrier in this example was used toconjugate to a free thiol on the protein and peptide in the examplesbelow. The synthesis is outlined in FIG. 3.

Briefly, compound Vd (20 mg, 0.044 mmol, 1 equiv.) and compound VIIa(Quanta Biodesign cat. #10140, with n=25 where n is the number ofrepeating CH₂CH₂O units, 44 mg, 0.0346 mmol, 0.8 equiv.) were dissolvedin anhydrous dichloromethane (1 mL). Triethylamine (22.0 mg, 31 μl, 0.22mmol, 5 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL), washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was purified by silica gel (10 g) flashchromatography. The column was eluted with 1-10% MeOH/dichloromethane.Fractions containing pure product were combined together and evaporatedon a rotavap, while maintaining the temperature below 30° C. The samplewas dried under a stream of nitrogen to afford compound VIIb as a browngum (33 mg, 56%) (R_(f) 0.20, silica gel, 10% methanol indichloromethane). ¹H NMR analysis of the isolated material confirmed itsidentity.

Compound VIIb (31 mg, 0.018 mmol, 1 equiv.), N-hydroxysuccinimide (6.3mg, 0.055 mmol, 3 equiv.), and EDCI (8.6 mg, 0.045 mmol, 2.5 eq.) weredissolved in anhydrous THF (2 mL). Triethylamine (7.4 mg, 10 μL, 0.073mmol, 4 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL) and washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was dried under a stream of nitrogen toafford compound VII, VitD-(3)-PEG_(2k)-NHS, as a brown gum (38.6mg, >100%) (R_(f) 0.25, silica gel, 10% methanol in dichloromethane). ¹HNMR analysis of the isolated material confirmed its identity and purity.

Example 5 Preparation and Characterization of Apelin Conjugated toNon-Hormonal Vitamin D

In this example, apelin conjugated to the VitD-(25)-PEG_(2K)-maleimidecarrier generated in Example 1 and the VitD-(3)-PEG_(2K)-aldehydegenerated in Example 2 to apelin imparted a significantly longerhalf-life for apelin. The resulting conjugated molecule may be a usefultherapeutic for the treatment of heart disease, pulmonary hypertension(e.g. pulmonary arterial hypertension or pulmonary venous hypertension),other cardiovascular diseases, or diabetes.

Synthesis of VitD-(25)-PEG_(2K)-C-Apelin

An apelin-13 derivative with a N-terminal cysteine residue (C-apelin)was synthesized by Biopeptek, Inc. (Malvern, Pa., SEQ ID NO:16).Conjugation with the carrier was accomplished by mixing a thiol-reactivemoiety [VitD-(25)-PEG_(2K)-maleimide from Example 1] dissolved in DMSOat 5 mg/mL with the apelin peptide containing a free cysteine at aconcentration of 5 mg/mL in PBS buffer with 1 mM EDTA in a molar ratioof 1.4:1 carrier to peptide. The reaction was allowed to proceed for 1hour at room temperature. The conjugated peptide,VitD-(25)-PEG_(2K)-apelin, was separated from unreacted components byion exchange chromatography. Conjugation and purity was confirmed bySDS-PAGE. The conjugates were then buffer exchanged to PBS and filtersterilized using a 0.22 micron filter for use in the animal study.

Synthesis of VitD-(3)-PEG_(2K)-Apelin:

The apelin-13 peptide was purchased from Bachem (Torrance, Calif., Cat.NO. H-4566). Conjugation between the aldehyde on the carrier and anamine moiety on the peptide was carried out at low pH in order to favorreaction with the N-terminal amine of the peptide. The amine-reactivecarrier [Vitamin D-(3)-PEG_(2K)-aldehyde from Example 2] dissolved inDMSO at 5 mg/mL was mixed with the apelin peptide at a concentration of5 mg/mL in dH₂O in a molar ratio of 3:1 carrier to peptide with a finalconcentration of 50 mM NaOAc pH=5 and 25 mM NaCNBH₃. The reaction wasallowed to proceed overnight at 4° C. The conjugated peptide,VitD-(3)-PEG_(2K)-apelin, was separated from unreacted components by ionexchange chromatography. Conjugation and purity was confirmed bySDS-PAGE.

Activity of Apelin Constructs in Cell-Based APJ Receptor Assay:

Unmodified apelin-13, VitD-(25)-PEG_(2K)-C-apelin, andVitD-(3)-PEG_(2K)-apelin were submitted to Multispan, Inc. (Hayward,Calif.) for determination of bioactivity. Multispan's functional apelinassay uses HEK293T cells expressing the receptor for apelin, APJ(Multispan catalog #: C1196). The assay measures apelin inhibition offorskolin-stimulated cAMP production. A comparison of the functionalactivity of apelin with the two modified peptides is shown in FIG. 4.The curves were fit with a four parameter logistic function in order todetermine the EC₅₀ values. The EC₅₀ value for apelin was approximately 6nM, with the modified apelin derivatives being 3-4 fold higher, withinthe observed error for this experiment. Thus, unmodified and modifiedapelin have substantially the same activity.

Pharmacokinetic Properties of Apelin and Apelin Conjugates:

Four groups of four rats each were injected intravenously withapelin-13, VitD-(25)-PEG_(2K)-C-apelin, or Vitamin D-(3)-PEG_(2K)-apelinat 0.1 mg/kg. Plasma samples were taken at 5 min, and 0.5, 1, 2, 4, 8and 24 hr and analyzed for the presence of apelin by quantitative ELISA(Phoenix Pharmaceuticals, Burlingame, Calif., Cat. No. EK-057-23). Theapelin conjugates showed dramatically improved pharmacokinetic profileswhen compared to unmodified apelin (FIG. 5). Unmodified apelin decayedto near-background levels within 5 minutes of injection.VitD-(25)-PEG_(2K)-C-apelin showed improved pharmacokinetic propertieswhen compared to unmodified apelin. Surprisingly, however,VitD-(3)-PEG_(2K)-apelin showed significantly improved pharmacokineticproperties when compared to the other apelin molecules, includingVitD-(25)-PEG_(2K)-C-apelin. This demonstrated that conjugation of thecarrier to the C3 position of vitamin D provides further improvement toconjugation at the C25 position.

The decay rates of the carrier-conjugates are complex and probablyrepresent a combination of slower renal clearance and protection fromprotease degradation. A pharmacokinetic analysis of the data was thenperformed using WinNonLin (PharSight) and GraphPad (Prism). Importantly,the AUC of the native apelin-13 peptide was 0.4 ng*hr/mL, VitaminD-(25)-PEG_(2K)-apelin was 85 ng*hr/mL, and Vitamin D-(3)-PEG_(2K) was328 ng*hr/mL. Therefore, the bioavailability improvements of the C25 andC3 carriers were 213-fold and 820-fold, respectively.

Example 6 Preparation and Characterization of Ghrelin Conjugated toNon-Hormonal Vitamin D

Synthetic ghrelin peptides are listed in Table 1A. Wild type (wt)peptides were purchased from Bachem [Torrence, Calif., Catalog # H-4864(human) and H-4862 (rat)], and custom sequences were synthesized byBiopeptek (Malvern, Pa.). Custom sequences include peptides where theoctanoylated serine (Oct-S, also known as O-octanoyl-serine) at positionthree is replaced by octanoylated 2,3-diaminopropionic acid (Oct-Dap,also known as Nβ□octanoyl-2,3-diaminopropionic acid), tyrosine (Y), ortryptophan (W).

Oct-S is rapidly deacylated in vivo by esterases; the deacylated form ofghrelin no longer activates the GHS-R receptor. The Oct-Dap, tryptophan,and tyrosine derivatives should maintain their activity since they arenot subject to deacetylation by esterases. The vitamin D-PEG-maleimidecarrier, as described in Examples 1 and 3, was selected to beproportional in size to a 2-3 kDa peptide so that conjugation might notsignificantly affect the bioactivity. Conjugation with the carrier wasaccomplished by mixing a thiol-reactive moiety[VitD-(25)-PEG_(2K)-maleimide from Example 1, or compound VI:VitD-(3)-PEG_(2K)-maleimide from Example 3, or PEG_(2K)-maleimide fromSigma-Aldrich #731765, also known as poly(ethylene glycol) methyl ethermaleimide] dissolved in DMSO at 5 mg/mL with the ghrelin peptidecontaining a free cysteine at a concentration of 5 mg/mL in PBS bufferwith 1 mM EDTA in a molar ratio of 1.4:1 carrier to peptide. Thereaction was allowed to proceed for 1 hour at room temperature. Theconjugated peptide was separated from unreacted components by ionexchange chromatography. Conjugation and purity was confirmed bySDS-PAGE. Rat ghrelin peptides (rGhrelin), human ghrelin peptides(hGhrelin) and the ghrelin-carrier conjugates were then buffer exchangedto PBS and filter sterilized using a 0.22 micron filter for use in theanimal study.

TABLE 1A Spe- SEQ. Name Sequence cies ID wt Oct- GS(Oct-S) human 2hGhrelin FLSPEHQRVQQRKESKKPPAKLQPR Oct- GS(Oct-S) human 3 hGhrelin-CFLSPEHQRVQQRKESKKPPAKLQPRC Dap- GSS(Oct-Dap) human 4 hGhrelin-CFLSPEHQRVQQRKESKKPPAKLQPRC GSY- GSYFLSPEHQRVQQRKESKKPPAKL human 5hGhrelin-C QPRC wt Oct- GS(Oct-S) rat 6 rGhrelinFLSPEHQKAQQPKESKKPPAKLQPR Oct- GS(Oct-S) rat 7 rGhrelin-CFLSPEHQKAQQPKESKKPPAKLQPRC Dap- GS(Oct-Dap) rat 8 rGhrelin-CFLSPEHQKAQQPKESKKPPAKLQPRC GSW- GSWFLSPEHQKAQQPKESKKPPAKLQ rat 9rGhrelin-C PRCPharmacokinetics of Ghrelin Conjugates

The pharmacokinetics of ghrelin conjugates were examined in SpragueDawley rats. The conjugates were: wt Oct-hGhrelin and Oct-hGhrelin-Cconjugated to VitD-(25)-PEG_(2K)-maleimide; Dap-hGhrelin-C conjugated toeither VitD-(25)-PEG_(2K)-maleimide or compound VI:VitD-(3)-PEG_(2K)-maleimide from Example 3; and PEG_(2K)-maleimide.Briefly, 0.1 mg/kg of each molecule was injected separately into therats by intravenous (iv) or subcutaneous (sc) injection. Samples ofplasma were collected at 5 mins (iv only), 30 mins, 1 hr, 2 hrs, 4 hrs,8 hrs, and 24 hrs. Protease inhibitors and HCl (final concentration 0.05N) were added to the plasma samples, which were then immediately frozen.Samples were analyzed using commercial ELISA kits validated foranalyzing either total rGhrelin (acylated+non-acylated) or activerGhrelin (acylated only) from rat plasma (Millipore, Cat. #EZRGRT-91Kand #EZRGRA-90K). The results show significant differences in thepharmacokinetic profiles of hGhrelin and the hGhrelin-carrier conjugates(FIGS. 6-8).

FIG. 6A shows that the pharmacokinetic profile of intravenously-injectedghrelin was improved when conjugated to vitamin D at the C25 and C3positions. Active and total ghrelin levels were analyzed because theOct-Dap modification protected the protein from deacylation, whereas thenative Oct-Ser containing peptides were rapidly deacylated.Wt-Oct-hGhrelin alone or conjugated to the VitD-(25)-PEG_(2k)-maleimidecarrier, as well as Dap-hGhrelin conjugated to either theVitD-(25)-PEG_(2k)-maleimide carrier or the VitD-(3)-PEG_(2k)-maleimidecarrier were injected intravenously into Sprague-Dawley rats at 0.1mg/kg. Ghrelin concentrations in plasma samples were analyzed by ELISAfor either total ghrelin (acylated+non-acylated) or active ghrelin(acylated only) in duplicate. The average value from three animals wasplotted on the semi-log graph. The octanoylated Dap residue wasresistant to degradation.

Both the C25 and C3 conjugates showed significant improvements in thepharmacokinetic profiles. The C3 conjugate, however, showed the bestpharmacokinetic profile. Compared to unmodified wt Oct-hGhrelin, thePEG_(2K)-(25)-VitD carrier provided significant half-life extension toboth Oct-hGhrelin-C and Dap-hGhrelin, although the former is rapidlydeacylated to its inactive form. The PEG_(2K)-(3)-VitD carrier providedeven more half-life extension when compared to the PEG_(2K)-(25)-VitDcarrier.

FIG. 7 compares active and total ghrelin levels subcutaneously injectedinto rats. It demonstrates that the PEG_(2K)-(25)-VitD andPEG_(2K)-(3)-VitD carriers provided significant half-life extension andimprovements in bioavailability. The carrier modified at the C3 positionof vitamin D was superior. The Oct-Dap modification provided resistanceto deacylation, however, some degree of deacylation occurred followingsubcutaneous injection that was not observed with intravenous delivery.

FIG. 8 compares the pharmacokinetic profiles for Dap-hGhrelin-Cconjugated to PEG_(2k)-maleimide, VitD-(25)-PEG_(2k)-maleimide, andVitD-(3)-PEG_(2k)-maleimide carrier. The samples were injected eitherintravenously or subcutaneously. Plasma samples were analyzed for totalGhrelin (acylated+non-acylated) levels by ELISA. While both the C25 andC3 conjugates showed significant improvement over the PEGylated ghrelin,the C3 conjugate showed the most improved bioavailability andpharmacokinetic properties. The 2 kDa PEG scaffold alone had somehalf-life extending properties for intravenously-injected ghrelin. Itwas ineffective, however, for modifying the pharmacokinetic propertiesof subcutaneously-injected ghrelin. In contrast, modification of ghrelinwith PEG_(2K)-(25)-VitD or PEG_(2K)-(3)-VitD carrier resulted insignificantly longer half-lives upon intravenous injection.Additionally, bioavailability was improved following subcutaneousinjection. The carrier modified at the C3 position of vitamin D wassuperior to the carrier modified at the C25 position.

Assessment of the Receptor Binding Activity of a Ghrelin Peptide-Carrier

In some embodiments, the activity of the ghrelin peptide, whenconjugated to a carrier, are substantially the same as unmodifiedpeptides. Ghrelin and VitD-PEG-ghrelin were compared for receptorbinding and activation of a ghrelin receptor (agonist activity) using acell-based receptor agonist assay: HEK293T cells stably expressing humanghrelin receptor (GHS-R, Multispan Cat. No. C1197b) were monitored forincreased intracellular calcium upon exposure to the test compoundsusing the Screen Quest™ Fluo-8 No Wash kit (AAT Bioquest, Cat. No.36315) on a FLIPR 384 instrument (Molecular Devices, Cat. Nos. FLIPR and0200-6072). EC₅₀ values for unconjugated and conjugated ghrelin weredetermined and compared. The EC₅₀ value for rGhrelin was 1.9 nM. TheEC₅₀ value for Dap-rGhrelin-C-PEG_(2k)-(3)-VitD was 20.5 nM. The EC₅₀value for GSW-rGhrelin-C-PEG_(2k)-(3)-VitD was 3.9 nM. Thus, conjugationof ghrelin to vitamin D resulted in substantially the same receptoractivity as the unmodified ghrelin peptide.

Pharmacokinetics of Ghrelin Conjugates with Multiple Dosing

The long-term pharmacokinetic profiles of two ghrelin conjugates,Dap-rGhrelin-C-PEG_(2k)-(3)-VitD and GSW-rGhrelin-C-PEG_(2k)-(3)-VitD,were compared to unmodified wt Oct-rGhrelin at various doses. Each dosewas delivered by two subcutaneous injections separated by 48 hrs withblood collection as follows: t=0 (before first dose), 0.5, 1, 2, 4, 8,24, 32, 48 (before second dose), 48.5, 49, 50, 52, 56, 72, 80, and 96hours. The doses examined were 5 and 0.5 mg/kg (wt Oct-rGhrelin), 0.5and 0.1 mg/kg (Dap-rGhrelin-C-PEG_(2k)-(3)-VitD, and 0.5 and 0.1 mg/kg(GSW-rGhrelin-C-PEG_(2k)-(3)-VitD). Pefabloc SC (Sigma-AldrichCat#76309) was added to collected plasma samples at 1 mg/ml and theplasma was immediately frozen until the levels of ghrelin were analyzedusing a rat/mouse ghrelin (total) ELISA kit (Millipore, Cat.#EZRGRT-91K). Both carrier-modified ghrelin conjugates show greatlyimproved pharmacokinetic profiles compared to unmodified ghrelin (FIG.6B). At the highest dose of ghrelin conjugates (0.5 mg/kg), measurablelevels of the conjugates were observed 72 hours after the secondinjection, whereas the same dose of unmodified ghrelin returned tobaseline levels within 6 hrs. The concentration profiles as a functionof time for 0-48 hours were analyzed with Kinetica software(ThermoFisher) using an extravascular, non-compartmental analysis. Areaunder the curve (AUC) was calculated using the trapezoidal (linear rule)method. Results for the 0.5 mg/kg doses are given in Table 1B. Theconjugates achieved higher peak concentrations (Cmax) and possessedslower elimination times than unmodified ghrelin leading to largeincreases in the calculated AUC (30-50 fold). Calculated t_(1/2) valuesincreased from 0.6 hours to around 10 hours with the VitD-(3) carrier, a17-fold improvement.

TABLE 1B Dose C_(max) T_(max) AUC t_(1/2) MRT Compound (mg/kg) (ng/ml)(hr) hr*(mg/ml) (hr) (hr) wt Oct- 0.5 319 0.5 373 0.59 1.06 rGhrelinDap- 0.5 1096 4 19,332 9.57 13.64 rGhrelin-C- PEG_(2k)-(3)- VitD GSW-0.5 547 4 10,921 10.26 15.05 rGhrelin-C- PEG_(2k)-(3)- VitDGhrelin Conjugates for Treating Cachexia and Other Weight LossConditions

As an animal model of cancer cachexia, rats were implanted with YoshidaAH130 ascites hepatoma cells. After implantation of the tumor cells, along-lived ghrelin conjugate, Dap-rGhrelin-C-PEG_(2k)-(3)-VitD, wasdelivered either every day, or every other day, at various doses.Unmodified ghrelin was delivered by constant infusion via a subcutaneousosmotic pump. Dap-rGhrelin-C-PEG_(2k)-(3)-VitD was also compared tosimilar subcutaneous doses of ghrelin andOct-rGhrelin-C-PEG_(2k)-(3)-VitD (a long-lived but not constitutivelyactive ghrelin). See Table 1C for a complete listing of compound dosing,where “ghrelin”=wt Oct-rGhrelin,“Dap-VitD”=Dap-rGhrelin-C-PEG_(2k)-(3)-VitD, and“Oct-VitD”=Oct-rGhrelin-C-PEG_(2k)-(3)-VitD.

TABLE 1C Dose Group N Tumor Agent (mg/kg) Route Schedule 1 10 Nonevehicle — sc qd to end 2 10 None Dap-VitD 0.1 sc qd to end 3 10 Yesvehicle — sc qd to end 4 10 Yes Ghrelin 1.25/day sc osmotic 6 days ofpump infusion 5 10 Yes Ghrelin 0.1 sc qd to end 6 10 Yes Ghrelin 0.5 scqd to end 7 10 Yes Oct-VitD 0.1 sc qd to end 8 10 Yes Dap-VitD 0.1 sc qdto end 9 10 Yes Dap-VitD 0.5 sc qod to end (days 1, 3, 5)

Nine week old female Wistar rats (Crl:WI, Charles River Labs.) with abody weight (BW) range of 185.1-266.3 g, on Day 1 were used. YoshidaAH-130 rat hepatoma cells were propagated in vivo in the rats. On theday of the first inoculation, the hepatoma cells were thawed, washed toremove freezing medium, resuspended in PBS, and injectedintraperitoneally (ip) into each rat. For subsequent in vivo passages,hepatoma cells were harvested from the ascites fluid during log phasegrowth and resuspended in phosphate-buffered saline (PBS) at 2×10⁷cells/mL. On Day 0, animals in Groups 3-9 (n=10/group) each received anip injection of 0.2 mL of the cell suspension. Groups 1 and 2 were notinoculated.

On Day 1, rats were placed into nine groups of ten animals and weretreated in accordance with the protocol in Table 1C. Doses for Groups1-3 and 5-8 were delivered subcutaneously (sc), every day (qd). Dosesfor Group 4 were delivered via subcutaneous osmotic pump (Model 2001, 1μl/hr). Doses for Group 9 were delivered every other day (qod). Groups 1and 3 received vehicle, Groups 2 and 8 received Dap-VitD at 0.1 mg/kg,and Group 9 received Dap-VitD at 0.5 mg/kg. Group 4 received ghrelin at1.25 mg/kg/day via s.c. osmotic pump, and Groups 5 and 6 receivedghrelin at 0.1 and 0.5 mg/kg. Group 7 received Oct-VitD at 0.1 mg/kg.All doses for Groups 1-3 and 5-9 were delivered in a dose volume of 1mL/kg and were dosed to the individual body weight of each animal. Bodyweight (BW) was recorded for each animal on Days 1-6. The significanceof differences among the means of the normalized bodyweight values forthe treatment groups was determined by using an unpaired t-test(GraphPad). In tumor-inoculated groups, when an animal presented withless than 1.0 mL of ascites fluid, engraftment failure was assumed andthe data for that animal was not used.

The change in body weight during the course of treatment is shown inFIG. 9. It shows a plot of average body weight for each group (n=7-10)normalized to 100 percent on Day 1. The untreated group with implantedtumors (Group 3) lost body weight over the course of six days. Incontrast, all of the treated groups with tumors (Groups 4-9) displayedincreased body weight approaching that of healthy animals without tumors(Groups 1 and 2). On Day 4, the average body weight of each group wasstatistically different than Group 3 (p≦0.05 vs. Group 3). On Day 4, ofall the groups bearing tumors, the group that received a constantinfusion of ghrelin (Group 4) had the highest weight, followed closelyby the group receiving Dap-rGhrelin-C-PEG_(2k)-(3)-VitD every other dayby subcutaneous injection (Group 9).

These data show that ghrelin conjugated to vitamin D can effectivelyincrease the body weight in subjects. This can be used to reverse thewasting effects of cachexia and other weight loss disorders. Thesuperior pharmacokinetic profiles provided by the vitamin D carriersprovide for more flexibility in dosing times and amounts.

Example 7 Preparation of Insulin Coupled to Non-Hormonal Vitamin D atthe C25 and C3 Positions

In this example, the VitD-(25)-PEG_(2k)-NHS was conjugated to humaninsulin comprising the A chain (SEQ ID NO:11) and B chain (SEQ ID NO:12)to prepare a therapeutic for treating diabetes. The insulin A chaincontains a cys6-cys11 intra-chain disulfide linkage. The cys7 on the Achain is linked to cys7 of the B chain by an inter-chain disulfidelinkage. The cys20 on the A chain is linked to cys19 of the B chain,also by an interchain disulfide linkage. Insulin (Sigma Aldrich, St.Louis, Mo., Catalog #12643) was resuspended in a 1:1 mixture of DMSO and1M HEPES+0.85% NaCl, pH=8 at a concentration of 5 mg/ml.VitD-(25)-PEG_(2k)-NHS carrier dissolved in DMSO at a concentration of 5mg/ml 1.4 to 4 molar equivalents relative to insulin was added. Thefinal concentration of insulin was brought to 1 mg/ml in dH₂O and thereaction was allowed to proceed for 1 hour at room temperature. Theinsulin conjugates were confirmed by SDS-PAGE.

In this example, the VitD-(3)-PEG_(1.3k)-NHS is conjugated to humaninsulin to prepare a therapeutic for treating diabetes. Insulin (SigmaAldrich, St. Louis, Mo., Catalog # I2643) is resuspended in a 1:1mixture of DMSO and 1M HEPES+0.85% NaCl, pH=8 at a concentration of 5mg/ml. VitD-(3)-PEG_(1.3k)-NHS carrier is dissolved in DMSO at aconcentration of 5 mg/ml 1.4 to 4 molar equivalents relative to insulinwas added. The final concentration of insulin is brought to 1 mg/ml indH₂O and the reaction is allowed to proceed for 1 hour at roomtemperature. The insulin conjugates are confirmed by SDS-PAGE.

Pharmacokinetic experiments, in vitro bioactivity assays measuring theuptake of glucose by adipocytes, and evaluation in vivo of the bloodglucose lowering ability in diabetic rat models are performed asdescribed in EP2085406, incorporated herein by reference in itsentirety.

Example 8 Preparation of PTH Coupled to Non-Hormonal Vitamin D at the C3Position and the C25 Position

The VitD-(3)-PEG_(2K)-maleimide carrier (Compound VI from Example 3),the VitD-(3)-PEG_(2K)-aldehyde (Compound V from Example 2), and theVitD-(25)-PEG_(2K)-maleimide carrier (from Example 1), were conjugatedto PTH in order to extend the half-life of PTH, thereby making theconjugated molecule a potentially useful therapeutic for the treatmentof hypoparathyroidism and osteoporosis.

Synthesis of PTH-C-PEG_(2K)-(3)-VitD

A PTH derivative with a C-terminal cysteine residue was synthesized byBiopeptek, Inc. (Malvern, Pa., SEQ ID NO:17). Conjugation with thecarrier was accomplished by mixing the thiol-reactiveVitD-(3)-PEG_(2K)-maleimide carrier (Compound VI from Example 3)dissolved in DMSO at 5 mg/mL with the PTH peptide containing a freecysteine at a concentration of 5 mg/mL in PBS buffer with 1 mM EDTA in amolar ratio of 1.3:1 carrier to peptide. The reaction was allowed toproceed for 100 minutes at room temperature. The conjugated peptide,PTH-C-PEG_(2K)-(3)-VitD, was separated from unreacted components by ionexchange chromatography. Conjugation and purity was confirmed bySDS-PAGE. The conjugates were then buffer exchanged to PBS and filtersterilized using a 0.22 micron filter for use in the animal study.

Synthesis of VitD-(3)-PEG_(2K)-PTH:

The human PTH(1-34) peptide was purchased from Bachem (Torrance, Calif.,Catalog # H-4835, SEQ ID NO:10). Conjugation between the aldehyde on thecarrier and an amine moiety on the peptide was carried out at low pH inorder to favor reaction with the N-terminal amine of the peptide. Theamine-reactive VitD-(3)-PEG_(2K)-aldehyde carrier (Compound V fromExample 2) dissolved in DMSO at 5 mg/mL was mixed with the PTH(1-34)peptide at a concentration of 5 mg/mL in dH₂O in a molar ratio of 3:1carrier to peptide with a final concentration of 50 mM NaOAc pH=5 and 25mM NaCNBH₃. The reaction was allowed to proceed overnight at 4° C. Theconjugated peptide, VitD-(3)-PEG_(2K)-PTH, was separated from unreactedcomponents by ion exchange chromatography. Conjugation and purity wasconfirmed by SDS-PAGE.

Activity of PTH(1-34) Constructs in Cell-Based PTH1 Receptor Assay:

Unmodified PTH(1-34), PTH-C-PEG_(2K)-(3)-VitD, and VitD-(3)-PEG_(2K)-PTHwere submitted to Multispan, Inc. (Hayward, Calif.) for determination ofbioactivity. Multispan's functional PTH assay uses mammalian cellsexpressing the PTH1 receptor (Multispan Catalog #C1301). The assaymeasures agonist activity using calcium mobilization (Screen Quest™Fluo-8 No Wash kit, AAT Bioquest catalog #36315) and a cAMP assay (HTRFcAMP HiRange Kit, CisBio catalog #62AM6PEC). A comparison of thefunctional activity of PTH(1-34) vs the two modified peptides is shownin Table 2. The curves were fit with a four parameter logistic functionin order to determine the EC₅₀ values. The EC₅₀ values for PTH(1-34) andPTH-C-PEG_(2K)-(3)-VitD were very similar, while the EC₅₀ value forVitD-(3)-PEG_(2K)-PTH was approximately 10-20 fold worse. This showedthat conjugation to the C-terminus of PTH resulted in substantially thesame activity as unmodified PTH(1-34). Conjugation at the N-terminus ofPTH, however, interfered with its activity.

TABLE 2 Compound Calcium EC₅₀ cAMP EC₅₀ PTH(1-34) 18.8 nM 13.1 pMPTH-C-PEG_(2K)-(3)-VitD 14.0 nM 18.6 pM VitD-(3)-PEG_(2K)-PTH  126 nM 380 pMSynthesis of VitD-(25)-PEG_(2K)-C-PTH

A PTH derivative with a N-terminal cysteine residue was synthesized byBiopeptek, Inc. (Malvern, Pa., SEQ ID NO:18). Conjugation with thecarrier was accomplished by mixing the thiol-reactiveVitD-(25)-PEG_(2K)-maleimide carrier from Example 1 dissolved in DMSO at5 mg/mL with the PTH peptide containing a free cysteine at aconcentration of 5 mg/mL in PBS buffer with 1 mM EDTA in a molar ratioof 1:3:1 carrier to peptide. The reaction was allowed to proceed for 75minutes at room temperature. The conjugated peptide,VitD-(25)-PEG_(2K)-C-PTH, was separated from unreacted components by ionexchange chromatography. Conjugation and purity was confirmed bySDS-PAGE. The conjugates were then buffer exchanged to PBS and filtersterilized using a 0.22 micron filter for use in the animal study.

Synthesis of VitD-(3)-PEG_(2K)-C-PTH

A PTH derivative with a N-terminal cysteine residue was synthesized byBiopeptek, Inc. (Malvern, Pa., SEQ ID NO:18). Conjugation with thecarrier was accomplished by mixing the thiol-reactiveVitD-(3)-PEG_(2K)-maleimide carrier (Compound VI from Example 3)dissolved in DMSO at 5 mg/mL with the PTH peptide containing a freecysteine at a concentration of 5 mg/mL in PBS buffer with 1 mM EDTA in amolar ratio of 1.3:1 carrier to peptide. The reaction was allowed toproceed for 75 minutes at room temperature. The conjugated peptide,VitD-(3)-PEG_(2K)-C-PTH, was separated from unreacted components by ionexchange chromatography. Conjugation and purity was confirmed bySDS-PAGE. The conjugates were then buffer exchanged to PBS and filtersterilized using a 0.22 micron filter for use in the animal study.

Pharmacokinetics of PTH Conjugates

PTH conjugates show improved pharmacokinetics in Sprague Dawley ratswhen compared to free PTH. Unmodified PTH(1-34),VitD-(25)-PEG_(2K)-C-PTH, and VitD-(3)-PEG_(2K)-C-PTH were compared.Briefly, 0.1 mg/kg of each molecule was injected separately into therats (n=4) by subcutaneous (sc) injection. Samples of plasma werecollected at 0 hrs (pre-dose), 0.5, 1, 2, 4, 8, 12, 24, 32, 48, and 56hours then immediately frozen. Samples were analyzed for human PTH(1-34) by ELISA (Phoenix Pharmaceuticals Cat# EK-055-08). The resultsshow significant differences in the pharmacokinetic profiles of PTH andthe PTH-carrier conjugates (FIG. 10).

FIG. 10 shows that the pharmacokinetic profile ofsubcutaneously-injected PTH was improved when conjugated to vitamin D atthe C25 and C3 positions. The best pharmacokinetic profile was obtained,however, with conjugation to the C3 position. Pharmacokinetic parameterswere obtained by analyzing the data with Kinetica software(ThermoFisher). Reliable parameters for unmodified PTH could not beobtained due to the poor bioavailability of the wild-type peptide andbackground PTH signal from endogenous rat PTH. The half-life ofPTH(1-34), however, when dosed subcutaneously in rats has previouslybeen reported to be between 15 and 60 minutes (Frolick, Bone 33: 372-379(2003) and Satterwhite, Calcif Tissue Int. 87:485-492 (2010)).Therefore, the half-life of 2.2 hr for VitD-(25)-PEG_(2K)-C-PTH and 6.9hr for VitD-(3)-PEG_(2K)-C-PTH represent improvements of at least 2- and7-fold, respectively, compared to unmodified PTH. Likewise, the vitaminD-conjugates show improvements in bioavailability as indicated by the10-fold higher Cmax values and at least a 5-fold and 15-fold improvementin AUC values for the C25 and C3 conjugates, respectively.

Example 9 Preparation of an Antibody Coupled to Non-Hormonal Vitamin Dat the C3 Position

In this example, the VitD-(25)-PEG_(2k)-NHS carrier (described inWO2013172967) and the VitD-(3)-PEG_(1.3k)-NHS carrier (Compound VII)were conjugated to infliximab (Remicade®) in order to extend thehalf-life and bioavailability of the antibody. Remicade is used to treatCrohn's Disease, rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, and plaque psoriasis.

Infliximab (Remicade®), sold as a lyophilized powder with theappropriate salts (Hannah Pharmaceuticals), was resuspended to aconcentration of 10 mg/mL with water. VitD-(25)-PEG_(2k)-NHS or theVitD-(3)-PEG_(1.3k)-NHS carrier was resuspended at a concentration of 10mg/mL in DMSO. VitD-(3)-PEG_(1.3k)-NHS and the infliximab were thenmixed at a molar ratio of 5:1, 10:1, or 30:1 carrier to infliximab. Atherapeutic compound carrier conjugate of the invention typically has atleast 1 and could be between 1-10 carrier molecules individuallyattached to a therapeutic compound. By using an NHS version of thecarrier, more than one carrier can be attached to a therapeutic protein.This can be controlled by altering the molar ratio of carrier to targettherapeutic in the reaction. In this example, a target distribution of1-4 carriers was used. This was confirmed by testing two different molarratios and examining the resulting conjugates by mass spectrometry. C25carriers were conjugated to the antibody at a ratio of about between1-2:1. C3 carriers were conjugated to the antibody at a ratio of about1:1.

The infliximab and infliximab NHS-carrier conjugates were separated fromunconjugated carrier by use of a desalting column with a 40 kDa cutoff(Zeba Spin, Thermo Scientific). MALDI-TOF mass spectrometry was used tocalculate the intact mass of infliximab in the reactions. The resultsshow that unmodified infliximab had a mass predominantly of 149 kDa. Anaverage attachment of one to three of the VitD-(25)-PEG_(2k)-NHS carrierwas attached to the antibodies and one to two of theVitD-(3)-PEG_(1.3k)-NHS carrier was attached to the antibodies.

Example 10 Preparation and Characterization of GLP-1 Conjugated toNon-Hormonal Vitamin D

Synthetic GLP-1(7-37) peptide (SEQ ID NO: 19, hereafter referred to asGLP-1) was purchased from Bachem (Torrence, Calif., Catalog # H-9560),and GLP-1-C with an additional C-terminal cysteine residue (SEQ ID NO:20) was custom synthesized by Biopeptek (Malvern, Pa.). It wasconjugated to the Vitamin D-PEG-maleimide carrier as described inExamples 1 and 3. Conjugation was accomplished by mixing athiol-reactive moiety (VitD-(25)-PEG_(2K)-maleimide) from Example 1, orcompound VI (VitD-(3)-PEG_(2K)-maleimide) from Example 3 dissolved inDMSO at 10 mg/mL with the GLP-1-C peptide containing a free cysteine ata concentration of ˜1 mg/mL in PBS buffer with 1 mM EDTA in a molarratio of 1:3:1 carrier to peptide. The reaction was allowed to proceedfor 1 hour at room temperature. The conjugated peptide was separatedfrom unreacted components by ion exchange chromatography. Conjugationand purity was confirmed by SDS-PAGE. GLP-1 peptide and theGLP-1-carrier conjugates were then buffer exchanged to PBS.

Assessment of the Receptor Binding Activity of GLP-1 Peptide-Carrier

In some embodiments, the activity of the GLP-1 peptide, when conjugatedto a carrier, was about the same as unmodified peptides. GLP-1 andGLP-1-C-PEG_(2K)-VitD were compared for receptor binding and activationof GLP1R, the GLP-1 receptor, (agonist activity) using a cell-basedreceptor agonist assay: PathHunter® cells stably expressing human GLP-1receptor (GLP1R, DiscoveRx Corp., Fremont, Calif.) were monitored forrecruitment of γ-Arrestin upon exposure to the test compounds using thePathHunter® Detection reagent cocktail on a PerkinElmer Envisioninstrument with chemiluminescent signal detection. The EC₅₀ value forunconjugated GLP-1 was 0.82 μM. The EC₅₀ value forGLP-1-C-PEG_(2K)-(25)-VitD was 0.51 μM. The EC₅₀ value forGLP-1-C-PEG_(2K)-(3)-VitD was 0.52 μM. Thus, conjugation of GLP-1 tovitamin D results in about the same or better receptor activity as theunmodified GLP-1 peptide.

Example 11 Preparation and Characterization of FGF21 Conjugated toNon-Hormonal Vitamin D at the C3 and C25 Positions

A modified FGF21 was conjugated to the Vitamin D-PEG-maleimide carrieras described in Examples 1 and 3. As shown below, the FGF21-carriercomposition provided significantly improved pharmacokinetic propertieswhen compared to an unmodified FGF21. While both conjugates showed asignificant improvement over the unmodified FGF21, conjugation at the C3position showed significant improvement over conjugation at the C25position. Together, this example shows that an FGF21-VitD conjugate isan important therapeutic compound for the treatment of diseases thatwould benefit from FGF21 treatment, including diabetes.

FGF21 was expressed in E. coli, purified, and conjugated to the carrieras follows. A modified FGF21 with a free cysteine residue near the aminoterminus of FGF21 allowed site-specific coupling to the carrier. A 6-Histag (SEQ ID NO: 23) was added for ease of purification. The modifiedFGF21 coding sequence (SEQ ID NO: 21) was computationally codonoptimized for expression in E. coli. The gene was chemically synthesizedby DNA2.0 (Menlo Park, Calif.) and cloned into the IP-Free expressionvector pD441-SR that contains an IPTG-inducible T5 promoter and akanamycin resistance gene. The plasmid was transformed into Shuffle®Express Competent E. coli (New England BioLabs Cat. No: C3028H). Cellswere grown to mid-log phase at 30° C. and then induced for four hours at25° C. with 0.1 mM IPTG Cells were harvested, lysed, and the supernatantcollected. The FGF21 protein (SEQ ID NO: 22) was purified usingimmobilized metal affinity chromatography (IMAC) resin and polished byanion exchange chromatography.

Conjugation with the carrier was accomplished by mixing a thiol-reactivemoiety VitD-(25)-PEG_(2K)-maleimide from Example 1 or compound VI:VitD-(3)-PEG_(2K)-maleimide from Example 3. Briefly, the carriers weredissolved in DMSO at 10 mg/mL with the purified FGF21 protein containinga free cysteine in a molar ratio of 3:1 carrier to FGF21. The reactionwas allowed to proceed for 1 hour at room temperature. The conjugatedpeptide was separated from unreacted components by using a Zeba™ spindesalting column, 7K MWCO, according to the manufacturer's protocol(ThermoFisher Scientific Inc., Cat. No. 89882). Conjugation and puritywas confirmed by SDS-PAGE. FGF21, VitD-(25)-PEG_(2K)-FGF21, andVitD-(3)-PEG_(2K)-FGF21 were then buffer exchanged to PBS and filtersterilized using a 0.22 micron filter for use in the animal study.

Assessment of the Receptor Binding Activity of FGF21-Carrier Conjugates

In some embodiments, the activity of the FG21, when conjugated to acarrier, was substantially the same as unmodified protein. FGF21 and theVitD-PEG_(2K)-FGF21 conjugates were compared for receptor binding andactivation of FGFR1 using a cell-based receptor agonist assay(PathHunter® U2OS FGFR1-β-Klotho Functional Assay, DiscoveRx Corp.,Fremont, Calif., Cat. No. 93-0943C3). PathHunter® cells stably expresshuman FGF21 receptor (FGFR1). They were monitored for recruitment of theco-receptor, β-Klotho, following exposure to the test compounds usingthe PathHunter® Detection reagent cocktail on a PerkinElmer Envisioninstrument with chemiluminescent signal detection. The EC₅₀ value forunconjugated FGF21 was 0.16 μg/ml. The EC₅₀ value forVitD-(25)-PEG_(2K)-FGF21 was 0.13 μg/ml. The EC₅₀ value forVitD-(3)-PEG_(2K)-FGF21 was 0.40 μg/ml. Thus, the FGF21 conjugatesretained about the same receptor activity as the unmodified FGF21protein or better.

Pharmacokinetics of FGF21 Conjugates

The pharmacokinetics of FGF21 conjugates in Sprague Dawley rats weredetermined. Unmodified FGF21, VitD-(25)-PEG_(2K)-FGF21 andVitD-(3)-PEG_(2K)-FGF21 were compared. Briefly, 0.1 mg/kg of eachmolecule was injected separately into the rats (n=3) by subcutaneous(sc) injection. Samples of plasma were collected at 0 hrs (pre-dose),0.5, 1, 2, 4, 8, 24, 32, 48, and 56 hours and were immediately frozen.The samples were analyzed using commercial ELISA kits for human FGF21(Millipore Cat. No. EZHFGF21-19K). The results show significantdifferences in the pharmacokinetic profiles of unconjugated FGF21 andthe FGF21-carrier conjugates (FIG. 11). The pharmacokinetic profile ofsubcutaneously-injected FGF21 was improved when conjugated to vitamin Dat the C25 and C3 positions. The best pharmacokinetic profile wasobtained, however, with conjugation to the C3 position. Thepharmacokinetic parameters were obtained by analyzing the data withKinetica software (ThermoFisher) and are listed in Table 3. Thehalf-life (t_(1/2)) of 5.3 hr for VitD-(25)-PEG_(2K)-FGF21 and 11.5 hrfor VitD-(3)-PEG_(2K)-FGF21 represent improvements of 4.1- and 8.8-fold,respectively, compared to unmodified FGF21. Likewise, the vitamin Dconjugates show improvements in bioavailability as indicated by theapproximately 2-fold higher Cmax values and a 7.2-fold and 12.0-foldimprovement in AUC values for the C25 and C3 conjugates, respectively.Improvements in the mean residence time (MRT) and terminal rate constant(Lz) values were also observed.

TABLE 3 ng/ml h h(ng/ml) 1/h h h Conjugation Cmax Tmax AUCtot Lz t1/2MRT None 59 2 228 .534 1.3 2.8 C25 115 2 1640 .131 5.3 7.9 C3  118 42727 .0604 11.5 18.9

Exemplary Sequences

(Apelin) SEQ ID NO: 1 QRPRLSHKGPMPF (human wt Oct-hGhrelin) SEQ ID NO: 2GS(Oct-S)FLSPEHQRVQQRKESKKPPAKLQPR (human Oct-hGhrelin-C) SEQ ID NO: 3GS(Oct-S)FLSPEHQRVQQRKESKKPPAKLQPRC (human Dap-hGhrelin) SEQ ID NO: 4GSS(Oct-Dap)FLSPEHQRVQQRKESKKPPAKLQPRC (human GSY-hGhrelin) SEQ ID NO: 5GSYFLSPEHQRVQQRKESKKPPAKLQPRC (rat wt Oct-rGhrelin) SEQ ID NO: 6GS(Oct-S)FLSPEHQKAQQPKESKKPPAKLQPR (rat Oct-rGhrelin) SEQ ID NO: 7GS(Oct-S)FLSPEHQKAQQPKESKKPPAKLQPRC (rat Dap-rGhrelin) SEQ ID NO: 8GS(Oct-Dap)FLSPEHQKAQQPKESKKPPAKLQPRC (rat GSW-rGhrelin) SEQ ID NO: 9GSWFLSPEHQKAQQPKESKKPPAKLQPRC (PTH (1-34)) SEQ ID NO: 10SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (human insulin A Chain) SEQ ID NO: 11GIVEQCCTSICSLYQLENYCN SEQ ID NO: 12: (human insulin B Chain)FVNQHLCGSHLVEALYLVCGERGFFYTPKT (human TNF-α) SEQ ID NO: 13MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDFSFISPFAQAVRSSSRTPSDKPVAHVVANPQAEGQFQWFNRRANAFFANGVEFRDNQFVVPSEGFYFIYSQVFFKGQGCPSTHVFFTHTISRIAVSYQTKVNFFSAIKSPCQRETPEGAEAKPWYEPIYFGGVFQFEKGDRFSAEINRPDYFDFAESGQVYFGIIAF(Vitamin D Binding Protein (DBP)) SEQ ID NO: 14MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQFVKEVVSFTEACCAEGADPDCYDTRTSAFSAKSCESNSPFPVHPGTAECCTKEGFERKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNI L(Vitamin D Binding Protein (DBP)) SEQ ID NO: 15TTTAATAATAATTCTGTGTTGCTTCTGAGATTAATAATTGATTAATTCATAGTCAGGAATCTTTGTAAAAAGGAAACCAATTACTTTTGGCTACCACTTTTACATGGTCACCTACAGGAGAGAGGAGGTGCTGCAAGACTCTCTGGTAGAAAAATGAAGAGGGTCCTGGTACTACTGCTTGCTGTGGCATTTGGACATGCTTTAGAGAGAGGCCGGGATTATGAAAAGAATAAAGTCTGCAAGGAATTCTCCCATCTGGGAAAGGAGGACTTCACATCTCTGTCACTAGTCCTGTACAGTAGAAAATTTCCCAGTGGCACGTTTGAACAGGTCAGCCAACTTGTGAAGGAAGTTGTCTCCTTGACCGAAGCCTGCTGTGCGGAAGGGGCTGACCCTGACTGCTATGACACCAGGACCTCAGCACTGTCTGCCAAGTCCTGTGAAAGTAATTCTCCATTCCCCGTTCACCCAGGCACTGCTGAGTGCTGCACCAAAGAGGGCCTGGAACGAAAGCTCTGCATGGCTGCTCTGAAACACCAGCCACAGGAATTCCCTACCTACGTGGAACCCACAAATGATGAAATCTGTGAGGCGTTCAGGAAAGATCCAAAGGAATATGCTAATCAATTTATGTGGGAATATTCCACTAATTACGGACAAGCTCCTCTGTCACTTTTAGTCAGTTACACCAAGAGTTATCTTTCTATGGTAGGGTCCTGCTGTACCTCTGCAAGCCCAACTGTATGCTTTTTGAAAGAGAGACTCCAGCTTAAACATTTATCACTTCTCACCACTCTGTCAAATAGAGTCTGCTCACAATATGCTGCTTATGGGGAGAAGAAATCAAGGCTCAGCAATCTCATAAAGTTAGCCCAAAAAGTGCCTACTGCTGATCTGGAGGATGTTTTGCCACTAGCTGAAGATATTACTAACATCCTCTCCAAATGCTGTGAGTCTGCCTCTGAAGATTGCATGGCCAAAGAGCTGCCTGAACACACAGTAAAACTCTGTGACAATTTATCCACAAAGAATTCTAAGTTTGAAGACTGTTGTCAAGAAAAAACAGCCATGGACGTTTTTGTGTGCACTTACTTCATGCCAGCTGCCCAACTCCCCGAGCTTCCAGATGTAGAGTTGCCCACAAACAAAGATGTGTGTGATCCAGGAAACACCAAAGTCATGGATAAGTATACATTTGAACTAAGCAGAAGGACTCATCTTCCGGAAGTATTCCTCAGTAAGGTACTTGAGCCAACCCTAAAAAGCCTTGGTGAATGCTGTGATGTTGAAGACTCAACTACCTGTTTTAATGCTAAGGGCCCTCTACTAAAGAAGGAACTATCTTCTTTCATTGACAAGGGACAAGAACTATGTGCAGATTATTCAGAAAATACATTTACTGAGTACAAGAAAAAACTGGCAGAGCGACTAAAAGCAAAATTGCCTGATGCCACACCCACGGAACTGGCAAAGCTGGTTAACAAGCACTCAGACTTTGCCTCCAACTGCTGTTCCATAAACTCACCTCCTCTTTACTGTGATTCAGAGATTGATGCTGAATTGAAGAATATCCTGTAGTCCTGAAGCATGTTTATTAACTTTGACCAGAGTTGGAGCCACCCAGGGGAATGATCTCTGATGACCTAACCTAAGCAAAACCACTGAGCTTCTGGGAAGACAACTAGGATACTTTCTACTTTTTCTAGCTACAATATCTTCATACAATGACAAGTATGATGATTTGCTATCAAAATAAATTGAAATATAATGCAAACCATAAAAAAAAAAAAAAAAAAAAAAA (C-Apelin) SEQ ID NO: 16CQRPRLSHKGPMPF (PTH-C) SEQ ID NO: 17 SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC(C-PTH) SEQ ID NO: 18 CSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (GLP-1(7-37))SEQ ID NO: 19 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR G (GLP-1-C) SEQ ID NO: 20HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGC(nucleotide sequence encoding FGF21 protein with I3C substitution and His6tag(SEQ ID NO: 23)) SEQ ID NO: 21ATGCATCATCACCATCATCACCCGTGTCCAGATTCCTCTCCTTTATTGCAATTCGGTGGCCAAGTTCGTCAACGCTACCTGTATACCGACGACGCCCAGCAGACCGAAGCGCACCTTGAGATCCGTGAGGATGGTACGGTCGGTGGCGCAGCTGACCAAAGCCCGGAGAGCCTGCTGCAGTTGAAGGCCCTGAAACCGGGTGTTATCCAGATTCTGGGTGTGAAAACCAGCCGCTTTCTGTGCCAGCGTCCGGATGGCGCGCTGTACGGTAGCCTGCATTTCGACCCGGAAGCGTGCTCTTTTCGCGAGCTGCTGCTGGAAGATGGCTATAACGTGTACCAAAGCGAAGCGCACGGTCTGCCGCTGCATCTGCCGGGTAATAAGAGCCCGCACCGCGATCCGGCACCGCGTGGTCCGGCTCGTTTCCTGCCGTTGCCGGGTCTGCCACCGGCGCTGCCGGAGCCGCCAGGCATTCTGGCACCGCAGCCGCCTGACGTCGGCAGCAGCGACCCGCTGTCCATGGTTGGTCCGAGCCAGGGCCGTAGCCCGTCGTATGC GAGCTGATAA(FGF21 protein with I3C substitution and His6 tag (SEQ ID NO: 23))SEQ ID NO: 22MHEIHREIREIPCPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

All publications and patent documents disclosed or referred to hereinare incorporated by reference in their entirety. The foregoingdescription has been presented only for purposes of illustration anddescription. This description is not intended to limit the invention tothe precise form disclosed. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed:
 1. A carrier-drug conjugate, comprising a targetinggroup that is a non-hormonal vitamin D that is not hydroxylated at thecarbon 1 position, conjugated at the carbon 3 position to a proteinhaving apelin activity comprising an amino acid sequence with at least a90% sequence identity to SEQ ID NO:1.
 2. The carrier-drug conjugate ofclaim 1, wherein said targeting group is conjugated to said protein viaa scaffold that is poly(ethylene glycol).
 3. A pharmaceuticalcomposition comprising a carrier-drug conjugate comprising a targetinggroup that is a non-hormonal vitamin D that is not hydroxylated at thecarbon 1 position, conjugated at the carbon 3 position via a scaffold toa protein having apelin activity comprising an amino acid sequence withat least a 90% sequence identity to SEQ ID NO:
 1. 4. The pharmaceuticalcomposition of claim 3 wherein said scaffold is poly(ethylene glycol).5. The pharmaceutical composition of claim 3, wherein said amino acidsequence of said protein is SEQ ID NO:1.
 6. The pharmaceuticalcomposition of claim 3, wherein said scaffold has a mass of about 2 kDa.7. A pharmaceutical composition, comprising: a) a protein having apelinactivity comprising an amino acid sequence with at least a 90% sequenceidentity to SEQ ID NO:1, b) a stably attached scaffold, c) a targetinggroup that is a non-hormonal vitamin D that is not hydroxylated at thecarbon 1 position, conjugated at the carbon 3 position, wherein afteradministration to a first test subject, said protein has a half lifemeasured by Enzyme Linked Immunosorbant Assay (ELISA) analysis of bloodsamples taken at a plurality of time points that is greater than a halflife of said protein administered to a second test subject without saidstably attached scaffold moiety and targeting group as measured by saidELISA analysis of blood samples taken at said plurality of time points.8. The pharmaceutical composition of claim 7, wherein saidadministration to said first and second subjects is accomplished bysubcutaneous injection.
 9. The pharmaceutical composition of claim 7,wherein said protein stably attached to said scaffold and targetinggroup retains about the same activity as said protein not stablyattached to said scaffold and targeting group as measured by afunctional assay.
 10. The pharmaceutical composition of claim 7, whereinsaid amino acid sequence of said protein is SEQ ID NO:1.
 11. Thepharmaceutical composition of claim 7, wherein the scaffold has a massof about 500 Da to 5,000 Da.
 12. The pharmaceutical composition of claim7, wherein said scaffold is approximately the same mass as the protein.13. A carrier-drug conjugate, comprising a targeting group that isvitamin D that is not hydroxylated at the carbon 1 position,non-releasably conjugated at the carbon 3 position to a protein havingapelin activity comprising an amino acid sequence with at least a 90%sequence identity to SEQ ID NO:1.
 14. The carrier-drug conjugate ofclaim 13, wherein said vitamin D is non-hormonal.
 15. The carrier-drugconjugate of claim 13, wherein said protein retains about the sameactivity as said protein not conjugated to said targeting group asmeasured by a functional assay.
 16. The carrier-drug conjugate of claim13, wherein said targeting group is conjugated to said protein via ascaffold that is poly(ethylene glycol).
 17. The carrier-drug conjugateof claim 13, wherein said scaffold is approximately the same mass as theprotein.